Compositions and methods for diagnosis and treatment of tumors

ABSTRACT

Based on the observation of the cooperation of osteopontin (OPN) and matrixmetalloproteinase-9 (MMP-9) in the promotion of the metastatic phenotype, therapies and diagnostic assays are disclosed for the treatment of a tumor that overexpresses OPN, such as hepatocellular carcinoma (HCC), for example metastatic HCC. In one example, methods of treating a tumor include administration of an agent that reduces cellular invasion resulting from the interaction between a fragment of OPN (OPN-5kD) generated by MMP-9 cleavage and CD44 receptor. Examples of such agents include fragments of OPN-5kD and antibodies specific for OPN-5kD. Therapeutic compositions are also provided that include such agents. Also provided are methods of diagnosing or prognosing a tumor, for example by detecting expression of OPN-5kD peptide or OPN-c mRNA in a biological sample obtained from the subject. Also provided are antibodies that specifically bind OPN-5kD.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 12/340,211 filed onDec. 19, 2008 now U.S. Pat. No. 7,803,380, which is acontinuation-in-part of International Patent Application No.PCT/US2007/071712 filed on Jun. 20, 2007, which claims priority fromU.S. Provisional Application No. 60/805,298 filed Jun. 20, 2006, whichis herein incorporated by reference.

FIELD

This application relates to methods of diagnosing and treating a tumorthat has increased expression of osteopontin (OPN), such as metastatichepatocellular carcinoma (HCC), as well as compositions and kits thatcan be used for such methods. Also provided are antibodies thatspecifically bind to a fragment of OPN(OPN-5kD) generated bymetalloproteinase-9 (MMP-9) cleavage.

BACKGROUND

Elucidating the molecular events that promote tumor cell invasioncontinues to be a challenge for the treatment and prevention ofhepatocellular carcinoma (HCC).

HCC is a highly aggressive carcinoma of the liver which has beenreported to occur world-wide in increasing numbers. Intra-hepaticmetastatic recurrences via the portal vein are the main cause of deathin HCC patients who have undergone partial hepatectomy or livertransplantation (Korn, World J. Gastroenterol., 7:777-8, 2001).Identifying the main ‘players’ and how they contribute to the tumor cellmetastatic cascade, for example at the early stages of cellularinvasion, can present opportunities for lessening the severity of HCCthrough new therapeutic interventions.

Osteopontin (OPN, SPP1) is a secreted multi-functional glycoproteinexpressed at high levels in tumors and the surrounding stroma ofnumerous cancers, including those of the liver, and is alternativelyspliced into at least 3 isoforms (OPN-a, OPN-b, OPN-c) (Butler, Ann.N.Y. Acad. Sci., 760:6-11, 1995; Coppola et al., Clin. Cancer Res.,10:184-90, 2004). Increased serum and plasma OPN levels (˜4-10 fold) areassociated with advanced stage lung, hepatic, breast, colon, andprostate carcinomas (Oates et al., Invasion Metastasis, 17:1-15, 1997;Fedarko et al., Clin. Cancer Res., 7:4060-6, 2001; Singhal et al., Clin.Cancer Res., 3:605-11, 1997). OPN expression can predict high grade,late stage and early recurrence HCC (Pan et al., Cancer, 98:119-27,2003) and is highly correlated with tumor recurrence and decreasedpatient survival following orthotopic liver transplantation (Wang,Hepatology, 42(4), Suppl. 1:391 A, 2005). Prominent OPN levels have beendetected in metastatic HCC tumor cells at the leading edge ofpseudopodia and filopodia (Suzuki et al., J. Bone Miner. Res.,17:1486-97, 2002) and in macrophages at the tumor-stroma interface(Senger et al., Ann. N.Y. Acad. Sci., 760: 83-100, 1995). A correlationbetween OPN mRNA expression and primary HCC tumor metastasis has beenshown (Ye et al., Nat. Med., 9: 416-423, 2003). Cytoplasmic OPN wasdetected in vascularized regions of primary HCC tumors but not in normalliver. In addition, a neutralizing antibody to OPN decreased pulmonarysecondary lesions in nude mice and inhibited tumor cell invasion.

Local and extra-hepatic HCC tumor cell invasion is associated withextensive matrix remodeling, angiogenesis, and hepatocyte injury(McKenna et al., Am. J. Surg., 183: 588-94, 2002). The members of thezinc-dependent endopeptidase family of matrix metalloproteinases (MMPs)catabolize extracellular matrix components (Theret et al., Hepatology,34:82-88, 2001; Liaw and Crawford, Braz. J. Med. Biol. Res., 32:805-812,1999). Each MMP contains a catalytic and pro-peptide regulatory domainand a variable number of carboxy-terminal hemopoexin-like structuraldomains and are broadly divided into subclasses based on substrateactivity. The two gelatinases (MMP-2 and MMP-9) play roles in tumorinvasion and angiogenesis and participate in cancer progression inseveral neoplasias (Turpeenniemi-Hujanen, Biochimie, 87:287-97, 2005;Hanemaaijer et al., Int. J. Cancer, 86:204-7, 2000; Scorilas et al., Br.J. Cancer, 84:1488-96, 2001). Active MMP-9 enzymatically cleavesproteins of the basement membrane (such as collagens type IV, V, VII, X,and XIV) and can be detected at the invasive front of HCC (Kaneyoshi etal., Clin. Cancer Res., 7:4027-32, 2001). A substantial increase inMMP-9 mRNA levels in HCC primary metastatic tumors has been observed (Yeet al., Nat. Med. 9:416-23, 2003), which is consistent with indicationsof HCC tumor malignancy and MMP-9 abundance (Ashida et al., Am. J.Pathol., 149:1803-11, 1996; Wei et al., Hunan. Yi. Ke. Da. Xue. Xue.Bao., 28:212-6, 2003).

Stromelysin-1 (MMP-3) and matrilysin (MMP-7) are reported to cleave OPNat residues 166 and 210 (Agnihotri et al., J. Biol. Chem., 276:28261-7,2001). MMP-3/-7 digested OPN fragments increase AsPC-1 and HeLa tumorcell adhesion via cell surface integrin receptors and MMP-3 cleaved OPNcan increase mouse peritoneal macrophage cell migration. The thrombincoagulation factor also cleaves OPN at residue 168, resulting in twofragments of similar molecular weight (˜28-30 kD) can be detected in theserum and plasma of patients with cancer (Senger et al., Cancer Res.,48:5770-4, 1988). Thrombin-cleaved OPN can mediate increased tumor andmacrophage cell adhesion and migration via exposure of theamino-terminal reactive RGD sequence and binding to cell surfaceintegrins, namely the vitronectin receptor αVβ3, although interactionswith αVβ1, αVβ5, α4β1, and α9β1 have been described (Sodek et al., Crit.Rev. Oral Biol. Med., 11:279-303, 2000; Wai and Kuo, J. Surg. Res.,121:228-41, 2004; Weber, Biochim. Biophys. Acta, 1552:61-85, 2001).Conversely, the COOH-terminal thrombin-cleaved fragment has beenproposed to induce macrophage migration primarily through CD44 receptors(Weber et al., J. Leukoc. Biol., 72:752-61, 2002).

SUMMARY

Methods of treating a tumor that overexpresses osteopontin (OPN), suchas a tumor that overexpresses OPN (for example OPN-c) and MMP-9, as wellas diagnosing and prognosing such tumors, are provided. Examples of suchtumors include but are not limited to cancers of the liver, breast,colon, and prostate. The disclosed treatment, diagnostic, and prognosticmethods can be used in combination or individually.

The inventors have determined that MMP-9 mediates OPN proteolyticcleavage into several fragments including OPN-5kD (such as SEQ ID NO:4), which is a ligand for CD44 receptor. The interaction of OPN-5kD andthe CD44 receptor increases HCC tumor cell invasion. The inventors haveidentified several fragments of OPN-5kD that significantly reduce HCCtumor cell invasion, which may be achieved by disrupting the interactionbetween OPN-5kD and CD44 receptor. Based on this observation, newmethods of treating such tumors are disclosed, for example by usingshort invasive-blocking peptides targeted to the effects of OPN-5kD. Theinventors have also identified monoclonal antibodies that bind toOPN-5kD which can be used to significantly reduce HCC tumor cellinvasion by disrupting the interaction between OPN-5kD and the CD44receptor. Therefore, new methods of treating such tumors are disclosed,for example by administering therapeutically effective amounts of one ormore peptides or antibodies that reduce or inhibit the effects ofOPN-5kD.

Methods of treating a tumor in a subject are provided, such as a tumorthat overexpresses OPN, MMP-9, or both. A particular example of such atumor is HCC. In one example, the method includes administering to thesubject a therapeutically effective amount of an agent that decreasescellular invasion of a tumor cell effected by the interaction of OPN-5kDand the CD44 receptor, such as a peptide fragment of OPN-5 kD, or anantibody that binds to OPN-5kD, thereby treating the tumor. A particularexample of OPN-5kD sequence is provided in SEQ ID NO: 4. However, oneskilled in the art will appreciate that variants of this sequence (suchas polymorphisms) can retain OPN-5kD activity (such as the ability tobind to CD44 receptor with high affinity). For example, a variantOPN-5kD sequence may include at least one amino acid deletion,substitution, addition, or combinations thereof, such as 1-5conservative amino acid substitutions, while retaining the ability tostimulate cellular invasion upon interaction with the CD44 receptor.

Exemplary therapeutic peptides can include at least one peptidesequence, or a plurality of peptide sequences, wherein each peptidefragment of OPN-5kD includes at least 5 contiguous amino acids ofOPN-5kD and in some examples is no more than 50 amino acids. Forexample, the peptide can be a composition that includes one or morepeptides consisting of the amino acid sequence shown in SEQ ID NO: 5, 6,7, or 8. Exemplary therapeutic antibodies can include a singlemonoclonal antibody or an equivalent specific binding agent the binds toone epitope (e.g., amino acids 30-36 of SEQ ID NO: 4) or multipleantibodies that bind to more than one epitope in OPN-5kD. If desired,therapeutically effective amounts of one or more additional therapeuticagents, such as an anti-neoplastic chemotherapeutic agent, can beadministered to the subject. In particular examples, treatment includesreducing cellular invasion by a tumor, such as reducing or preventingmetastasis of a tumor. In some examples, treating the tumor prolongssurvival time of the subject (for example by at least 2 months, at least6 months, or even at least 12 months).

Also provided by the present disclosure are therapeutic compositionsthat can be used to treat a tumor, such as a tumor that overexpressesOPN (such as overexpresses OPN and MMP-9), for example HCC. Suchcompositions can be used in the therapeutic methods provided herein. Inone example, the composition includes one or more peptide fragments ofOPN-5kD. In a specific example, the composition includes one or morepeptides consisting of the amino acid sequence of SEQ ID NO: 5, 6, 7, or8. In another example the therapeutic compositions include antibodiesthat specifically bind to OPN-5kD. The disclosed compositions caninclude one or more pharmaceutically acceptable carriers, as well asadditional therapeutic agents (such as other anti-neoplastic agents).Such therapeutic compositions can also be part of a kit, such as a kitthat includes one more other anti-neoplastic agents (such as IL-2,IL-12, GM-CSF, a chemotherapeutic agent, or combinations thereof).

Methods of diagnosing and prognosing a tumor (such as a tumor thatoverexpresses OPN, MMP-9, or both) are provided. In some examples, suchmethods are performed prior to the treatment methods described herein.However, such methods can also be used independently of the disclosedtreatment methods. In particular examples, the method includesdetermining if the OPN-5kD fragment is present in the subject, forexample by detecting OPN-5kD in the subject's serum. For example, asample from the subject that includes peptides (such as serum) iscontacted with an agent that specifically binds to OPN-5kD (such as anantibody that specifically binds to an epitope of OPN-5kD), then it isdetermined whether the agent specifically bound to proteins in thesample. If specific binding of the agent to OPN-5kD in the sample isdetected, this indicates that the subject has a tumor (such as HCC), hasa poor prognosis (for example because this indicates that the tumor hasmetastasized), or both. Such a method provides a non-invasive means fordiagnosis and prognosis of a tumor, such as metastatic HCC. In anotherexample, the method includes contacting a sample from the subject thatincludes nucleic acid molecules (such as a tumor sample) with an agentthat permits detection of OPN-c nucleic acid molecules (such as cDNA ormRNA), then determining a relative amount of OPN-c nucleic acidmolecules in the sample. For example, if increased OPN-c mRNA expressionis detected in non-cancerous tissue adjacent to the tumor or in thetumor itself (or both), this indicates that the subject's tumor hasincreased metastatic potential. Similar methods can be used to measureOPN-c protein expression, as an alternative to OPN-c nucleic acidmolecule expression.

Also provided are antibodies that specifically bind OPN-5kD, such as anantibody that binds to an epitope sequence within SEQ ID NO: 4, or afragment thereof such as any of SEQ ID NOS: 5-8. A specific epitope isamino acids 30-36 of SEQ ID NO: 4. Such antibodies are useful fordetection and treatment of tumors and can be part of a kit. Diagnostickits can include other agents to permit detection of the antibody, suchas a labeled secondary antibody. A specific example of such antibodiesis the OPN-5kD monoclonal antibody 5 kd106-13 D. Thus, the 5 kd106-13 Dantibody, chimeric form or humanized form thereof or functional fragmentthereof can be used to detect OPN-5kD and OPN-expressing tumors, as wellas treat such tumors. Chimeric forms of 5 kd106-13 D, humanized forms of5 kd106-13 D, and functional fragments of 5 kd106-13 D, are alsodisclosed. The 5 kd106-13 D antibody, chimeric form, humanized form orfunctional fragment of these antibodies can be conjugated to an effectormolecule, such as a detectable marker, a therapeutic agent, or a toxin.Kits that contain the monoclonal antibody 5 kd106-13 D, a chimeric form,or a humanized form thereof or functional fragments thereof are alsodisclosed. Nucleic acid molecules encoding the monoclonal antibody 5kd106-13 D, a chimeric form or humanized form thereof or functionalfragments thereof are also disclosed. A hybridoma that produces 5kd106-13 D is also provided.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A are two plots showing that mRNA expression of OPN (left) andMMP-9 (right) correlate with HCC tumor metastasis. NM=non-metastatic(n=11), M=metastatic (n=14), MM=metastatic intra-hepatic lesion (n=10).All data were normalized to mRNA levels detected in the normal tissueimmediately adjacent to the tumor samples (T/N). Statistical analysiswas performed using Mann Whitney non-parametric t-tests (α=0.05).

FIG. 1B is a graph showing OPN and MMP-9 expression level correlation asdetermined using linear regression analysis after plotting values withineach patient case.

FIG. 2A is a digital image of a Western blot showing a time-dependentMMP-9-cleavage of recombinant human OPN (˜65 kD) (lanes 2-6). MMPinhibitors were added to separate reactions and analyzed after a 1-hourdigestion at 37° C.: EDTA (10 mM), MMP-9 inhibitor I (2 μM) and TIMP-1(0.1 μM) (lanes 8, 10, 11 respectively). Undigested OPN (lane 1), OPNexposed to thrombin cleavage (0.05 U) (lane 7) and TIMP-1 alone lanes(9) were included as controls.

FIG. 2B is a graph showing the kinetics of the MMP-9 specific OPNcleavage quantified by densitometric analysis of the three bands markedby arrows in FIG. 2A and presented as a fraction of starting values atTime=0.

FIG. 3A is a schematic drawing showing the expression vectors generatedto determine the activity level of OPN fragments.

FIG. 3B is a digital image of a Western blot showing expression of thefour constructs in FIG. 3A in HEK 293 cells.

FIG. 4A is a digital image of a Western blot (left) and a bar graphshowing densitometric analysis of OPN-5kD expression in FIG. 4A (right),demonstrating that endogenous OPN protein levels correlate with HCC cellline metastatic potential. Data were adjusted relative to Hep3B levels(=1) following normalization to beta-actin levels.

FIG. 4B is a digital image of a protein gel showing gelatinase activityrelative to pro-MMP-9, active MMP-9, and active MMP-2 standards.

FIG. 4C is a bar graph showing the percent cell invasion for three HCCcell lines. Data are presented as the mean percent cell invasion andstandard deviation following normalization to fluorescence readingscorresponding to cells migrating through uncoated control membranes.

FIG. 5A is a bar graph showing that MMP-9-cleaved OPN increased HCC celladhesion relative to uncleaved OPN. Student un-paired t-tests were usedto compare cleaved versus un-cleaved OPN adhesion (α=0.05).

FIG. 5B is a bar graph showing adhesion of HCC cells in response to fourOPN constructs. Student un-paired t-tests were used to compare mediaversus OPN adhesion at each time point (α=0.05).

FIG. 5C is a graph showing the effect of different length OPN moleculeson migration of SMMC-7721. Data are presented relative tovector-transfected cell controls at each of the time points. * denotessignificance p<0.017.

FIG. 6A is a bar graph showing that the OPN-5kD fragment inducedreproducible increases in cellular invasion. Data are presented as themean percent cell invasion and standard deviation followingnormalization to fluorescence readings corresponding to cells migratingthrough uncoated control membranes. Un-paired student's t-tests wereused to compare mean and standard deviation values (α=0.05).

FIG. 6B is a bar graph showing the effect of the presence of blockingantibodies to the integrin αVβ3 and CD44 receptors on the invasion ofHCC cells transiently expressing OPN full-length or OPN-5kD. Un-pairedstudent's t-tests were used to compare mean and standard deviationvalues (α=0.05).

FIG. 6C is a bar graph showing the effect of varying concentrations ofthe OPN-5kD peptide on the invasion of HCC cells. Un-paired student'st-tests were used to compare mean and standard deviation values of mediaversus treated cells (α=0.05).

FIGS. 7A and B are bar graphs showing that some 10-mer peptides caninhibit OPN-5kD induced HCC cellular invasion. Data were normalized touncoated membrane chamber values and adjusted by media control values(=1). (A) Un-paired student's t-tests were used to compare mean andstandard deviation values of OPN-5kD peptide versus each of the 10-merpeptides (α=0.05). (B) Significance of the small peptide inhibition ofthe OPN-5kD peptide increased cellular invasion was tested usingun-paired student's t-tests (α=0.05). * denotes significance (A) p<0.037and (B) p<0.002.

FIGS. 8A and 8B are plots showing the OPN splice variants expressed in(A) tumor or (B) non-cancerous tissue. Data are shown followingnormalization to 18S rRNA (Applied Biosystems, CA) and relative to anormal liver tissue reference pool (n=8).

FIG. 9 is a digital image of a western blot showing detection of OPN-5kDwith the monoclonal antibody 5 kd106-13 D.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and one letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 is a cDNA sequence for a human osteopontin-a.

SEQ ID NO: 2 is the protein encoded by SEQ ID NO: 1.

SEQ ID NO: 3 is a cDNA sequence for a human osteopontin-c.

SEQ ID NO: 4 is an amino acid sequence of OPN-5kD.

SEQ ID NO: 5 is the p3 fragment of OPN-5kD.

SEQ ID NO: 6 is the p6 fragment of OPN-5kD.

SEQ ID NO: 7 is the p7 fragment of OPN-5kD.

SEQ ID NO: 8 is a fragment of OPN-5kD.

SEQ ID NO: 9 is the protein encoded by SEQ ID NO: 3.

SEQ ID NO: 10 is a unique reporter sequence for OPN-a.

SEQ ID NO: 11 is a unique reporter sequence for OPN-c.

SEQ ID NOS: 12-15 show exemplary light chain frameworks of human MAbLEN.

SEQ ID NOS: 16-19 show exemplary heavy chain frameworks of human MAb21/28 CL.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a peptide”includes single or plural peptides and is considered equivalent to thephrase “comprising at least one peptide.” The term “or” refers to asingle element of stated alternative elements or a combination of two ormore elements, unless the context clearly indicates otherwise. As usedherein, “comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements. AllGenbank Accession numbers are incorporated by reference (the sequenceavailable on Dec. 15, 2008).

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs.

HCC: hepatocellular carcinoma

IL-2: interleukin-2

MMP-9: matrix metalloproteinase-9

OPN: osteopontin

Administration: To provide or give a subject an agent, such as acomposition that includes a peptide fragment of OPN-5kD, such as one ormore of SEQ ID NOS: 5-8, or OPN-5kD specific antibodies, by anyeffective route. Exemplary routes of administration include, but are notlimited to, oral, injection (such as subcutaneous, intramuscular,intradermal, intraperitoneal, and intravenous), sublingual, rectal,transdermal (e.g., topical), intranasal, vaginal and inhalation routes.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen. For example, OPN-5kD specificantibodies include those that specifically bind to the OPN-5kD fragmentantigen or an epitope thereof (e.g., EELNGAY, amino acids 30 to 36 ofSEQ ID NO: 4). Antibodies are composed of a heavy and a light chain,each of which has a variable region, termed the variable heavy (V_(H))region and the variable light (V_(L)) region. Together, the V_(H) regionand the V_(L) region are responsible for binding the antigen recognizedby the antibody. Includes polyclonal antibodies, monoclonal antibodies,and fragments thereof.

This includes intact immunoglobulins and the variants and portions ofthem well known in the art, such as Fab′ fragments, F(ab)′₂ fragments,single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies (for example, humanized murineantibodies), heteroconjugate antibodies (such as, bispecificantibodies). See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H.Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDRs inthree-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds an antigen of interest hasa specific V_(H) region and the V_(L) region sequence, and thus specificCDR sequences. Antibodies with different specificities (due to differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected, or a progeny thereof.Monoclonal antibodies are produced by methods known to those of skill inthe art, for instance by making hybrid antibody-forming cells from afusion of myeloma cells with immune spleen cells. Monoclonal antibodiesinclude humanized monoclonal antibodies. In some example a monoclonalantibody is the monoclonal antibody 5 kd106-13 D, which is specific forthe epitope EELNGAY (amino acids 30 to 36 of SEQ ID NO: 4).

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs or SDRs (which generally confer antigen binding) fromanother species, such as a murine antibody that specifically binds anepitope of the OPN-5kD fragment. Most typically, chimeric antibodiesinclude human and murine antibody domains, generally human constantregions and murine variable regions, murine CDRs and/or murine SDRs. Insome examples a chimeric antibody includes the SDRs or CDRs from themonoclonal antibody 5 kd106-13 D. In one example, a chimeric antibody isa hybrid protein composed of the variable or antigen-binding domain froma mouse antibody and the constant or effector domain from a humanantibody (such as an antibody that recognizes an epitope of OPN-5kD),although other mammalian species can be used, or the variable region canbe produced by molecular techniques. Methods of making chimericantibodies are well known in the art, for example, see U.S. Pat. No.5,807,715.

A “humanized antibody” is an immunoglobulin including a human frameworkregion and one or more CDRs from a non-human or SDRs (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” An exemplary donor isCDRs from monoclonal antibody 5 kd106-13 D. In one embodiment, all theCDRs are from the donor immunoglobulin in a humanized immunoglobulin.Constant regions need not be present, but if they are, they aresubstantially identical to human immunoglobulin constant regions, suchas at least about 85-90%, such as about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDRs, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. The acceptor framework of a humanizedimmunoglobulin or antibody can have a limited number of substitutions byamino acids taken from the donor framework. Humanized or othermonoclonal antibodies can have additional amino acid substitutions whichhave substantially no effect on antigen binding or other immunoglobulinfunctions. Exemplary conservative substitutions are described above (seealso U.S. Pat. No. 5,585,089). Humanized immunoglobulins can beconstructed by means of genetic engineering, for example, see U.S. Pat.Nos. 5,225,539 and 5,585,089.

Binding affinity: Affinity of an antibody for an antigen, such as theaffinity of an antibody (e.g., monoclonal antibody 5 kd106-13 D) for anOPN-5kD peptide fragment or epitope thereof. In one example, affinity iscalculated by a modification of the Scatchard method described byFrankel et al., Mol. Immunol., 16:101-106, 1979. In another example,binding affinity is measured by an antigen/antibody dissociation rate.In yet another example, a high binding affinity is measured by acompetition radioimmunoassay. In several examples, a high bindingaffinity is at least about 1×10⁻⁸ M. In other example, a high bindingaffinity is at least about 1.5×10⁻⁸, at least about 2.0×10⁻⁸, at leastabout 2.5×10⁻⁸, at least about 3.0×10⁻⁸, at least about 3.5×10⁻⁸, atleast about 4.0×10⁻⁸, at least about 4.5×10⁻⁸, or at least about5.0×10⁻⁸ M.

In a particular example, a functional variant of an OPN-5kD peptideretains a similar binding affinity for an antibody than the bindingaffinity of the native OPN-5kD peptide (such as SEQ ID NO: 4) for thesame antibody.

Cancer: Malignant neoplasm that has undergone characteristic anaplasiawith loss of differentiation, increased rate of growth, invasion ofsurrounding tissue, and is capable of metastasis.

CD44 receptors: A family of cell-surface adhesion molecules found onboth normal and malignant cell types that can mediate cell-matrix andcell-cell interactions. CD44 receptors have been associated withincreased inflammation and metastasis. Based on the results shownherein, it is proposed that OPN-5kD is a ligand for CD44 receptors, andthis interaction enhances cellular invasion, for example of HCC cells.

Chemotherapeutic agent: In cancer treatment, refers to theadministration of one or a combination of compounds to kill or slow thereproduction of rapidly multiplying cells. Chemotherapeutic agentsinclude anti-neoplastics known by those skilled in the art, including,but not limited to: 5-fluorouracil (5-FU), azathioprine,cyclophosphamide, antimetabolites (such as Fludarabine), antineoplastics(such as Etoposide, Doxorubicin, methotrexate, and Vincristine),carboplatin, cis-platinum and the taxanes, such as taxol, monoclonalantibodies such as Avastin or Herceptin, and growth pathway inhibitorssuch as Gleevac. In particular examples, such chemotherapeutic agentsare administered in combination with a therapy that reduces cellularinvasion (for example before, during or after administration of atherapeutic amount of one or more fragments of OPN-5kD that include atleast 5 contiguous amino acids of SEQ ID NO: 4, such as SEQ ID NOS: 5-8,or OPN-5kD specific antibodies).

Complementarity Determining Region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of animmunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3and H-CDR1, H-CDR2, H-CDR3, respectively. By definition, the CDRs of thelight chain are bounded by the residues at positions 24 and 34 (L-CDR1),50 and 56 (L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain arebounded by the residues at positions 31 and 35b (H-CDR1), 50 and 65(H-CDR2), 95 and 102 (H-CDR3), using the numbering convention delineatedby Kabat et al., (1991) Sequences of Proteins of Immunological Interest,5^(th) Edition, U.S. Department of Health and Human Services, PublicHealth Service, National Institutes of Health, Bethesda, Md. (NIHPublication No. 91-3242). CDRs contain the specificity determiningregions (SDRs) of the antibody. In some examples a CDR is a CDR from themonoclonal antibody 5 kd106-13 D.

Conservative substitution: One or more amino acid substitutions foramino acid residues having similar biochemical properties. Typically,conservative substitutions have little to no impact on the activity of aresulting polypeptide. For example, a conservative substitution in anOPN-5kD fragment (such as one or more conservative substitutions in anyof SEQ ID NOS: 5-8) ideally does not substantially affect the ability ofthe peptide to reduce invasion of an HCC cell. Methods that can be usedto determine the amount of invasion are disclosed herein (for example,see Examples 4-8). In another particular example, a conservativesubstitution in OPN-5kD (such as SEQ ID NO: 4) ideally does notsignificantly decrease the ability of OPN-5kD to specifically bind toCD44, and in some examples may not affect the ability of such aninteraction to stimulate cellular invasion. The interaction between anOPN-5kD variant containing one or more conservative amino acidsubstitutions and CD44 can be measured using methods known in the art,such as incubating a labeled OPN-5kD antibody with CD44receptor-expressing cells in the presence of OPN-5kD, wherein thepresence of detectable label indicates the presence of bound OPN-5kD tothe cells.

For example, an alanine scan can be used to identify which amino acidresidues in SEQ ID NOS: 4-8 can tolerate an amino acid substitution. Inone example, one or more conservative substitutions in SEQ ID NOS: 5-8ideally do not decrease the observed reduction of invasion by more than25%, for example not more than 20%, for example not more than 10%, whenan alanine, or other conservative amino acid (such as those listedbelow), is substituted for one or more native amino acids. In oneexample, one or more conservative substitutions in SEQ ID NO: 4 ideallydoes not reduce the observed interaction of SEQ ID NO: 4 with CD44receptor by more than 25%, for example not more than 20%, for examplenot more than 10%, when an alanine, or other conservative amino acid(such as those listed below), is substituted for one or more nativeamino acids.

In one example, one conservative substitution is included in thepeptide, such as a single conservative amino acid substitution in any ofSEQ ID NOS: 4-8. In another example, two conservative substitutions areincluded in the peptide (such as any of SEQ ID NOS: 4-8). In a furtherexample, three conservative substitutions are included in the peptide(such as any of SEQ ID NOS: 4-8). A peptide can be produced to containone or more conservative substitutions by manipulating the nucleotidesequence that encodes that polypeptide using, for example, standardprocedures such as site-directed mutagenesis or PCR. Alternatively, apeptide can be produced to contain one or more conservativesubstitutions by using standard peptide synthesis methods.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Examples of amino acids which may be substituted for anoriginal amino acid in a protein and which are regarded as conservativesubstitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glufor Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Glnfor His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leuor Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyrfor Trp; Trp or Phe for Tyr; and Ile or Leu for Val.

Further information about conservative substitutions can be found in,among other locations in, Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988) and in standard textbooks of genetics and molecular biology.

Decrease: To reduce the quality, amount, or strength of something.

In one example, a therapy decreases a tumor (such as the size or volumeof a tumor, the number of tumors, the metastasis of a tumor, orcombinations thereof), or one or more symptoms associated with a tumor,for example as compared to the response in the absence of the therapy.In a particular example, a therapy decreases the size or volume of atumor, the number of tumors, the metastasis of a tumor, or combinationsthereof, subsequent to the therapy, such as a decrease of at least 10%,at least 20%, at least 50%, or even at least 90%. Such decreases can bemeasured using the methods disclosed herein.

Deletion: The removal of a one or more nucleotides from a nucleic acidmolecule or the removal of one or more amino acids from a protein, theregions on either side being joined together.

Effector molecule: The portion of a chimeric molecule, for example achimeric molecule that includes an antibody (e.g., 5 kd106-13 D) orfragment thereof, that is intended to have a desired effect on a cell towhich the chimeric molecule is targeted. Effector molecules are alsoknown as an effector moieties (EM), therapeutic agents, or diagnosticagents, or similar terms.

Therapeutic agents include such compounds as nucleic acids, toxins,proteins, peptides, amino acids or derivatives, glycoproteins,radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleicacid therapeutic and diagnostic moieties include antisense nucleicacids, derivatized oligonucleotides for covalent cross-linking withsingle or duplex DNA, and triplex forming oligonucleotides.Alternatively, the molecule linked to a targeting moiety, such as anantibody, may be an encapsulation system, such as a liposome or micellethat contains a therapeutic composition such as a drug, a nucleic acid(such as an antisense nucleic acid), or another therapeutic moiety thatcan be shielded from direct exposure to the circulatory system. Means ofpreparing liposomes attached to antibodies are well known to those ofskill in the art. See, for example, U.S. Pat. No. 4,957,735; and Connoret al., Pharm. Ther. 28:341-365, 1985. Diagnostic agents or moietiesinclude radioisotopes and other detectable labels. Detectable labelsuseful for such purposes are also well known in the art, and includeradioactive isotopes such as ³²P, ¹²⁵I, and ¹³¹I, fluorophores,chemiluminescent agents, magnetic resonance imaging agents and enzymes.

Epitope: An antigenic determinant. An epitope is the particularstructure formed by chemical groups or peptide sequences in a moleculethat is antigenic, meaning that elicits a specific immune response. Anantibody specifically binds a particular antigenic epitope, for exampleon an OPN-5kD peptide fragment or portion thereof (e.g., EELNGAY, aminoacids 30 to 36 of SEQ ID NO: 4). Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, 6, 7, 8, 9 or 10 amino acids in a unique spatialconformation, such as this number of contiguous amino acids from OPN-5Kd (e.g., SEQ ID NO: 4). Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance. See, for example, “Epitope MappingProtocols” in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed(1996).

Framework Region: Amino acid sequences interposed between CDRs, andincludes variable light and variable heavy framework regions. Theframework regions serve to hold the CDRs of an antibody or fragmentthereof in an appropriate orientation for antigen binding.

Hepatocellular carcinoma (HCC): A malignant collection of abnormal anduncontrollably growing cells that derive from hepatocytes, theepithelial cells of the liver. Risk factors for development of livercancer include chronic infection with the hepatitis B or C virus andcirrhosis (scarring of the liver). HCC can be a primary tumor, an HCCtumor that has metastasized to another part of the body, or can resultfrom a metastasis to the liver from another part of the body (such asfrom colon, stomach, skin or ovarian cancer).

Immunoconjugate: A covalent linkage of an effector molecule to anantibody, such as 5 kd106-13 D antibody. The effector molecule can be adetectable label or a therapeutic molecule.

A “chimeric molecule” is a targeting moiety, such as a ligand or anantibody, conjugated (coupled) to an effector molecule. The term“conjugated” or “linked” refers to making two polypeptides into onecontiguous polypeptide molecule. In one embodiment, an antibody isjoined to an effector molecule (EM). In another embodiment, an antibodyjoined to an effector molecule is further joined to a lipid or othermolecule to a protein or peptide to increase its half-life in the body.The linkage can be either by chemical or recombinant means. In oneembodiment, the linkage is chemical, wherein a reaction between theantibody moiety and the effector molecule has produced a covalent bondformed between the two molecules to form one molecule. A peptide linker(short peptide sequence) can optionally be included between the antibodyand the effector molecule. Because immunoconjugates were originallyprepared from two molecules with separate functionalities, such as anantibody and an effector molecule, they are also sometimes referred toas “chimeric molecules.”

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope (e.g.,EELNGAY, amino acids 30 to 36 of SEQ ID NO: 4) to bind to that epitope(or cell expressing the epitope) to a detectably greater degree than,and/or to the substantial exclusion of, binding to substantially allother epitopes (or cells not expressing the epitope). Immunologicallyreactive conditions are dependent upon the format of the antibodybinding reaction and typically are those utilized in immunoassayprotocols or those conditions encountered in vivo. See Harlow & Lane(Antibodies: A Laboratory Manual. 1988) for a description of immunoassayformats and conditions. The immunologically reactive conditions employedin the methods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, pH) that are typical insidea living mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (e.g., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein, antibody, or cell) has been substantially separatedor purified away from other components, such as other components in thecell of the organism, or the organism itself, in which the componentoccurs, such as other chromosomal and extra-chromosomal DNA and RNA,proteins and cells. Nucleic acid molecules and proteins that have been“isolated” include nucleic acid molecules (such as DNA or RNA) andproteins purified by standard purification methods. The term alsoembraces nucleic acid molecules and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleic acidmolecules and proteins. For example, an isolated antibody is one that issubstantially separated from the animal or cells in which it wasgenerated, or from cell culture medium.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody (e.g., 5 kd106-13D) or a protein, to facilitate detection of that molecule. For example,the label can be capable of detection by ELISA, spectrophotometry, flowcytometry, or microscopy. Specific, non-limiting examples of labelsinclude fluorophores, chemiluminescent agents, enzymatic linkages,electron-dense compounds, haptens and radioactive isotopes. Methods forlabeling and guidance in the choice of labels appropriate for variouspurposes are discussed for example in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1998).

Linker peptide: A peptide within an antibody binding fragment (such asan Fv fragment, for example a 5 kd106-13 D antibody fragment) whichserves to indirectly bond the variable heavy chain to the variable lightchain. “Linker” can also refer to a peptide serving to link a targetingmoiety, such as a scFv, to an effector molecule, such as a cytotoxin ora detectable label.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an scFv. In the specific context, the terms include reference tojoining a ligand, such as an antibody moiety, to an effector molecule(“EM”). The linkage can be either by chemical or recombinant means.“Chemical means” refers to a reaction between the antibody moiety andthe effector molecule such that there is a covalent bond formed betweenthe two molecules to form one molecule.

Malignant: Cells which have the properties of anaplasia invasion andmetastasis.

Matrix metalloproteinase-9 (MMP-9): A member of the metalloproteinasefamily, also referred to as gelatinase B. It is called a gelatinasebecause it has a high affinity for digestion of denatured collagen I.MMP-9 can also cleave a variety of proteins including many components ofthe extracellular matrix such as collagens I, III, IV, and V, entactin,and elastin. MMP-9 is frequently up-regulated in cancer cells and alsoin the adjacent host tissues, and its expression by tumor cellscontributes to metastasis.

MMP-9 has a signal peptide that leads to its secretion, but the secretedmolecule is not itself enzymatically active. MMP-9 has a proregionadjacent to the signal peptide. The proregion contains a signaturesequence including an unpaired cysteine that interacts with the Zn²⁺that is complexed with three histidines to form the active site. Thisinteraction allows the proregion to act as a competitive inhibitor ofMMP-9. Hence, the proregion is cleaved and dissociated to allowenzymatic activity

MMP-9 has been cloned from a variety of organisms, and nucleic acid andprotein sequences are publicly available, for example from GenBank andEMBL (for example GenBank Accession Nos. CAC07541.1; NP_(—)038627.1;P14780.2; and NM_(—)004994.2). Human MMP-9 protein is Mr 92,000 althoughthe murine form is Mr 105,000 because of an insert of an additional 24amino acids.

Neoplasm: Abnormal growth of cells.

Normal cells: Non-tumor, non-malignant cells.

Osteopontin (OPN, SPP1): A secreted multi-functional phosphorylatedglycoprotein expressed at high levels in tumors and the surroundingstroma of numerous cancers, including those of the liver. OPN proteinscontain a functional Gly-Arg-Gly-Asp-Ser (GRGDS; amino acids 158-162 ofSEQ ID NO: 2) cell-binding sequence.

Several splice variants of OPN have been identified, including OPN-a(native sequence) and OPN-c (truncated sequence). OPN-c lacks exon 4 (27amino acids) in the NH₂-terminal region of the mature sequence. OPN-cincludes a transglutaminase reactive domain (Gly-X-Gly) which canmediate covalent homodimer cross-linking as well as heterodimerformation to other matrix components (such as fibronectin).

The term osteopontin includes any osteopontin gene, cDNA, mRNA, orprotein from any organism that retains OPN biological activity. OPNsequences are publicly available. For example, GenBank Accession Nos:D28759 (nucleic acid) and BAA05949 (protein) disclose human OPN-asequences, and GenBank Accession Nos: D2876 (nucleic acid) and BAA05951(protein) disclose human OPN-c sequences.

In certain examples, OPN has at least 80% sequence identity, for exampleat least 85%, at least 90%, at least 95%, or at least 98% sequenceidentity to a native OPN and retain OPN biological activity. In otherexamples, an OPN nucleic acid sequence has a sequence that hybridizesunder very high stringency conditions to a sequence set forth in GenBankAccession No. D28759 or D2876 and retains OPN activity.

OPN-5kD or OPN-5kD fragment: One of three osteopontin peptide fragmentsgenerated when exposed to MMP-9 (when run on a SDS gel this peptide canmigrate at approximately 10 kD, however, its predicted size based uponamino acid sequence is 5 kD). This peptide binds to the CD44 receptor,thereby enhancing cellular invasion of HCC cells. A particular exampleof an OPN-5kD sequence is provided in SEQ ID NO: 4. Methods ofdiagnosing a tumor (such as an OPN-c overexpressing tumor) by detectingOPN-5kD fragment, are provided herein. Peptide fragments of OPN-5kD areprovided herein, and can be used for example to treat anOPN-overexpressing tumor or to generate antibodies. Peptide fragments ofOPN-5kD include at least 5 contiguous amino acids of SEQ ID NO: 4).Specific examples of peptide fragments of OPN-5kD include peptides of 5to 60 or 5 to 50 amino acids in length (such as 5 to 25, 5 to 20, 5 to15, 6 to 12, 8 to 10, 10 to 44, 10 to 20, or 10 to 15 amino acids) andhave least 5 contiguous amino acids of an OPN-5kD sequence (e.g., SEQ IDNO: 4), such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 15, 16, 17, 18, 19,or 20 contiguous amino acids, such as 5 to 15 or 10 to 15 contiguousamino acids. Particular examples of peptide fragments of OPN5-kD areshown in SEQ ID NOS: 5-8 and amino acids 30-36 of SEQ ID NO: 4.

Peptide: A chain of amino acids of which is at least 4 amino acids inlength, regardless of post-translational modification (such asglycosylation or phosphorylation). In one example, a peptide is at least6 amino acids in length, such as at least 8, at least 9, at least 10, atleast 11, or at least 12 amino acids in length. In particular examples,a peptide is 4 to 30 amino acids in length, for example 5 to 25 aminoacids in length, 5 to 20 amino acids in length, 9 to 15 amino acids inlength, or 9 to 10 amino acids in length.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic agents, such as one or more compositions that include (1) apeptide fragment of OPN-5kD or (2) an OPN-5kD specific antibody (e.g., 5kd106-13 D).

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationscan include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate, sodium lactate, potassium chloride,calcium chloride, and triethanolamine oleate.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription. Promoters may be constitutive or inducible.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified peptideis one in which the peptide is more pure than the peptide in its naturalenvironment, such as within a cell.

In particular examples, purified OPN peptides or fragments thereof (suchas SEQ ID NOS: 4-8) refers to peptides that are at least 75% pure, atleast 80% pure, at least 90% pure, at least 95% pure, at least 97% pure,at least 98% pure, or at least 99% pure. The purity of a peptide can bemeasured using methods known in the art, such as by a Western blot.

Radiological agent: In cancer treatment, refers to the administration ofone or a combination of radioactive compounds to damage the DNA ofcells, thereby killing or slowing the reproduction of rapidlymultiplying cells. Exemplary methods of administering radiologicalagents to a subject include external beam radiotherapy (XBRT) orteletherapy, brachytherapy or sealed source radiotherapy, and unsealedsource radiotherapy. The radiological agents that can be administered toa subject in combination with the disclosed therapies that include apeptide of 5 to 50 amino acids in length that includes at least 5contiguous amino acids of OPN-5kD (such as at least 9 contiguous aminoacids of SEQ ID NO: 4) or OPN-5kD antibodies. Exemplary radiologicalagents include those known by those skilled in the art, such as ionizingradiation (for example x-rays and gamma rays).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can for example, be accomplished by chemicalsynthesis or by the artificial manipulation of isolated segments ofnucleic acids, such as by genetic engineering techniques. Similarly, arecombinant protein is one encoded for by a recombinant nucleic acidmolecule.

Sample: Includes biological samples that contain cells, genomic DNA,RNA, or proteins (or combinations thereof) obtained from a subject, suchas those present in peripheral blood (or a fraction thereof such asplasma or serum), urine, saliva, tissue biopsy, surgical specimen, fineneedle aspirate, and autopsy material. In a particular example, a sampleis obtained from a subject having or suspected of having a metastaticHCC.

Sequence identity: The identity between two or more nucleic acidsequences, or two or more amino acid sequences, is expressed in terms ofthe identity or similarity between the sequences. Sequence identity canbe measured in terms of percentage identity; the higher the percentage,the more identical the sequences are. Sequence similarity can bemeasured in terms of percentage similarity (which takes into accountconservative amino acid substitutions); the higher the percentage, themore similar the sequences are. Homologs or orthologs of nucleic acid oramino acid sequences possess a relatively high degree of sequenceidentity/similarity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. To compare two nucleic acid sequences,the options can be set as follows: −i is set to a file containing thefirst nucleic acid sequence to be compared (such as C:\seq1.txt); −j isset to a file containing the second nucleic acid sequence to be compared(such as C:\seq2.txt); −p is set to blastn; −o is set to any desiredfile name (such as C:\output.txt); −q is set to −1; −r is set to 2; andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two sequences: C:\B12seq c:\seq1.txt −j c:\seq2.txt−p blastn −o c:\output.txt −q −1 −r 2.

To compare two amino acid sequences, the options of B12seq can be set asfollows: −i is set to a file containing the first amino acid sequence tobe compared (such as C:\seq1.txt); −j is set to a file containing thesecond amino acid sequence to be compared (such as C:\seq2.txt); −p isset to blastp; −o is set to any desired file name (such asC:\output.txt); and all other options are left at their default setting.For example, the following command can be used to generate an outputfile containing a comparison between two amino acid sequences: C:\B12seqc:\seq1.txt −j c:\seq2.txt −p blastp −o c:\output.txt. If the twocompared sequences share homology, then the designated output file willpresent those regions of homology as aligned sequences. If the twocompared sequences do not share homology, then the designated outputfile will not present aligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1154 nucleotidesis 75.0 percent identical to the test sequence (1166=1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(that is, 15÷20*100=75).

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). Homologs are typically characterizedby possession of at least 70% sequence identity counted over thefull-length alignment with an amino acid sequence using the NCBI BasicBlast 2.0, gapped blastp with databases such as the nr or swissprotdatabase. Queries searched with the blastn program are filtered withDUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).Other programs use SEG. In addition, a manual alignment can beperformed. Proteins with even greater similarity will show increasingpercentage identities when assessed by this method, such as at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least98%, or at least 99% sequence identity. For example, a peptide havingsubstantial sequence identity to an OPN-5kD sequence can share at least80% sequence identity to SEQ ID NO: 4, such as at least 90% or at least95% sequence identity to SEQ ID NO: 4.

When aligning short peptides (fewer than around 30 amino acids), thealignment can be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). For example, when less than the entiresequence is being compared for sequence identity, homologs willtypically possess at least 75% sequence identity over short windows of10-20 amino acids, and can possess sequence identities of at least 85%,at least 90%, at least 95% or at least 98% depending on their identityto the reference sequence. For example, a peptide having substantialsequence identity to an OPN-5kD fragment can share at least 80% sequenceidentity to any of SEQ ID NOS: 5-8, such as at least 90%, at least 95%,at least 98%, or at least 99% sequence identity to SEQ ID NOS: 5-8.Methods for determining sequence identity over such short windows aredescribed at the NCBI web site.

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions, as described above. Nucleic acid sequences that do not showa high degree of identity may nevertheless encode identical or similar(conserved) amino acid sequences, due to the degeneracy of the geneticcode. Changes in a nucleic acid sequence can be made using thisdegeneracy to produce multiple nucleic acid molecules that all encodesubstantially the same protein. Such homologous nucleic acid sequencescan, for example, possess at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% sequence identitydetermined by this method. For example, a nucleic acid encoding anOPN-5kD sequence can have substantial sequence identity to a nativesequence if it shares at least 80% sequence identity to nucleotides566-697 of SEQ ID NO: 1, such as at least 90% or at least 95% sequenceidentity to nucleotides 566-697 of SEQ ID NO: 1. An alternative (and notnecessarily cumulative) indication that two nucleic acid sequences aresubstantially identical is that the peptide which the first nucleic acidsequence encodes is immunologically cross reactive with the peptideencoded by the second nucleic acid sequence.

One of skill in the art will appreciate that the particular sequenceidentity ranges are provided for guidance only; it is possible thatstrongly significant homologs could be obtained that fall outside theranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus an OPN-5kD specific binding agent is an agent thatbinds substantially to a OPN-5kD peptide or epitope thereof. In oneexample, the specific binding agent is an antibody that specificallybinds the OPN-5kD peptide.

The term “specifically binds” refers, with respect to an antigen such asOPN-5kD, to the preferential association of an antibody or otherspecific binding agent, in whole or part, to the antigen and not toother antigens. A certain degree of non-specific interaction can occurbetween a specific binding agent and a non-target antigen. Nevertheless,specific binding can be distinguished as mediated through specificrecognition of the antigen. Specific binding results in a significantassociation between the antibody (or other specific binding agent) andthe antigen than between the antibody and a non-antigen. Specificbinding typically results in greater than 2-fold, such as greater than5-fold, greater than 10-fold, or greater than 100-fold increase inamount of bound antibody or other specific binding agent to the antigenas compared to binding to a non-specific antigen.

The determination that a particular agent binds substantially only toOPN-5kD can be made using or adapting routine procedures. For example,western blotting can be used to determine that a specific binding agent,such as an antibody, binds substantially only to the protein (such assubstantially only binds OPN-5kD but not to other proteins, such asthose found in blood) (for example see Harlow and Lane, Antibodies: ALaboratory Manual. 1988). A variety of immunoassay formats areappropriate for selecting antibodies or other specific binding agentspecifically immunoreactive with a particular protein (such as OPN-5kD).For example, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals (such as laboratory or veterinarysubjects).

Therapeutically effective amount: An amount of a therapeutic agent (suchas an OPN-5kD specific antibody, or an OPN-5kD peptide fragment (such asa peptide of 5 to 50 amino acids in length that includes at least 5contiguous amino acids of OPN-5 kD), that alone, or together with one ormore additional therapeutic agents, induces the desired response, suchas treatment of a tumor that overexpresses OPN, such as a metastatic HCCtumor. In one example, it is an amount of an OPN-5kD specific antibody,or one or more peptide fragments of OPN-5kD needed to prevent or delaythe development of a tumor, prevent or delay the metastasis of a tumor,cause regression of an existing tumor, or treat one or more signs orsymptoms associated with a tumor, in a subject, such as a subject havingHCC. Ideally, a therapeutically effective amount provides a therapeuticeffect without causing a substantial cytotoxic effect in the subject.The preparations disclosed herein are administered in therapeuticallyeffective amounts.

In one example, a desired response is to decrease the size, volume, ornumber (such as metastases) of a tumor that overexpresses OPN, such asHCC. For example, the therapeutic compositions can decrease the size,volume, or number of tumors (such as HCC) by a desired amount, forexample by at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 50%, at least 75%, or even at least90%, as compared to a response in the absence of the composition.

The effective amount of a composition that includes an OPN-5kD specificantibody, or one or more peptide fragments of OPN-5kD, that isadministered to a human or veterinary subject will vary depending upon anumber of factors associated with that subject, for example the overallhealth of the subject. An effective amount of a composition can bedetermined by varying the dosage of the product and measuring theresulting therapeutic response, such as the regression of a tumor.Effective amounts also can be determined through various in vitro, invivo or in situ immunoassays. The disclosed therapeutic agents can beadministered in a single dose, or in several doses, as needed to obtainthe desired response. However, the effective amount of can be dependenton the source applied, the subject being treated, the severity and typeof the condition being treated, and the manner of administration.

In particular examples, a therapeutically effective dose of one or morepeptide fragments of OPN-5kD or an OPN-5kD antibody includes at least 1μg daily (such as 1-100 μg or 5-50 μg) if administered via injection, orat least 1 mg daily if administered topically (such as 1-100 mg or 5-50mg) of one or more peptides fragments of OPN-5kD or an OPN-5kD antibody.In particular examples, such daily dosages are administered in one ormore divided doses (such as 2, 3, or 4 doses) or in a singleformulation.

The disclosed compositions that include one or more peptide fragments ofOPN-5 kD or an OPN-5kD antibody can be administered alone, in thepresence of a pharmaceutically acceptable carrier, in the presence ofother therapeutic agents (such as other anti-neoplastic agents), orboth.

Transgene: An exogenous gene introduced into a cell.

Treating or treatment: Refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionrelated to a disease (such as a tumor, for example HCC). Treatment canalso induce remission or cure of a condition, such as a tumor. Inparticular examples, treatment includes preventing a tumor, for exampleby inhibiting the full development of a tumor, such as preventingdevelopment of a metastasis or the development of a primary tumor.Prevention does not require a total absence of a tumor.

Reducing a sign or symptom associated with a tumor (such as a tumor thatoverexpresses OPN, for example HCC) can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject (such as a subject having HCC which has not yet metastasized), areduction in severity of some or all clinical symptoms of the disease, aslower progression of the disease (for example by prolonging the life ofa subject having tumor), a reduction in the number of relapses of thedisease, an improvement in the overall health or well-being of thesubject, or by other parameters well known in the art that are specificto the particular tumor.

Tumor: A neoplasm.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity.

In one example, includes administering a therapeutically effectiveamount of a composition that includes an OPN-5kD specific antibody orone or more peptides fragments of OPN-5kD sufficient to allow thedesired activity. In particular examples the desired activity istreatment of a tumor, such as a tumor that expresses OPN, for exampleHCC.

Unit dose: A physically discrete unit containing a predeterminedquantity of an active material calculated to individually orcollectively produce a desired effect, such as a therapeutic effect. Asingle unit dose or a plurality of unit doses can be used to provide thedesired effect, such as treatment of a tumor, for example a metastaticHCC. In one example, a unit dose includes a desired amount of an agentthat decreases cellular invasion induced by OPN-5kD binding to CD44.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more therapeuticgenes and/or selectable marker genes and other genetic elements known inthe art. A vector can transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. A vector optionally includes materials to aidin achieving entry of the nucleic acid into the cell, such as a viralparticle, liposome, protein coating or the like. In a particularexample, a vector includes a nucleic acid molecule encoding one or moreof SEQ ID NOS: 4-8 or a fragment of SEQ ID NO: 4.

Methods of Treating a Tumor

It is shown herein that MMP-9 liberates an OPN-5kD fragment, and thatOPN-induced cellular invasion proceeds via binding of the OPN-5kD ligandto CD44 cell surface receptors. Without being bound to a particulartheory, it is proposed that exposure of a non-RGD cryptic binding siteregion of OPN by extracellular proteolytic cleavage increases binding tocell surface CD44 receptors and mediates an invasive response, which isdistinct and independent from integrin αVβ3 function. It is alsodemonstrated herein that small peptide fragments of OPN-5kD (for exampleany of SEQ ID NOS: 5-8) effectively decrease or inhibit cellularinvasion induced by OPN-5 kD. Without being bound to a particulartheory, it is proposed that these peptides structurally compete withOPN-5kD for available cell-surface CD44 docking sites, therebydecreasing cellular invasion. This observation also supports the use ofantibodies directed to epitopes within the OPN-5kD peptide (such asamino acids 30-36 of SEQ ID NO: 4) as therapeutics. Based on theseobservations, methods of treating a tumor that overexpresses OPN, MMP-9,or both, are disclosed. These methods include administering peptides,antibodies, or combinations thereof, which effectively decrease orinhibit cellular invasion.

A particular example of an OPN-5kD sequence is provided in SEQ ID NO: 4.However, one skilled in the art will appreciate that variants of SEQ IDNO: 4 (such as allelic variations that may be present betweenindividuals or organisms, which may include one or more substitutions,deletions, or insertions, or combinations thereof) can also be anOPN-5kD peptide, as long as such peptide retains the ability to bindCD44 receptor an stimulate cellular invasion of HCC cells (for exampleusing the methods described in Examples 4-7). In a particular example,an OPN-5kD sequence is 40 to 50 amino acids in length (such as 40-48,42-46, or 44 amino acids), and has at least 90% sequence identity to SEQID NO: 4, such as at least 92%, at least 95%, or at least 98% sequenceidentity to SEQ ID NO: 4. In one example, an OPN-5kD peptide is 40 to 50amino acids in length and includes 1-8 conservative amino acidsubstitutions in SEQ ID NO: 4, such as 1, 2, 3, 4, 5, 6, 7 or 8conservative amino acid substitutions in SEQ ID NO: 4.

Methods are disclosed herein for treating tumors, such as those thatoverexpress OPN, for example in combination with overexpression ofMMP-9. In one example, increased expression of OPN or MMP-9 can bedetected in serum or plasma obtained from a subject having such a tumor.For example, detection of OPN-5kD in the serum of a subject (for exampleat a level of at least twice that found in a subject not having atumor), detection of increased levels of OPN-c in the tumor (for examplerelative to expression of OPN-c in adjacent non-tumor cells, or relativeto expression of OPN-a in the tumor), or both, indicates that thesubject can benefit from the disclosed therapies. In some examples,subjects are initially screened to determine if they have increasedlevels of OPN-5kD in their serum, whether they have a tumor that hasincreased expression of OPN-c (for example relative to adjacentnon-tumor cells, or relative to expression of OPN-a), or combinationsthereof. For example, the diagnostic methods provided herein can be usedto screen subjects to determine if they are candidates for the disclosedtherapies.

In some examples, the tumor is treated in vivo, for example in amammalian subject, such as a human or veterinary subject. A tumor is anabnormal growth of tissue that results from excessive cell division. Aparticular example of a tumor is cancer. For example, the currentapplication is useful for the treatment (such as the prevention orreduction of metastasis) of tumors (such as cancers). Exemplary tumorsthat can be treated using the disclosed methods include, but are notlimited to: cancers of the liver, breast, colon, and prostate, includingmetastases of such tumors. For example, the tumor can be a tumor thatoverexpresses OPN (such as OPN-c), for example HCC, metastatic HCC, suchas an intra-hepatic metastasis or an extra-hepatic metastasis.

Treatment of a tumor, such as HCC or metastatic HCC, can includepreventing or delaying the development of the tumor in a subject (suchas preventing metastasis of a tumor), and also includes reducing signsor symptoms associated with the presence of such a tumor (for example byreducing the size or volume of the tumor or a metastasis thereof). In aspecific example, treatment includes reducing the growth of cells of thetumor, or even killing the tumor cells (for example by causing the cellsto undergo apoptosis). Such reduced growth can in some examples decreaseor slow metastasis of the tumor, or reduce the size or volume of thetumor. In one example, treatment of a tumor includes reducing theinvasive activity of the tumor in the subject, for example by reducingthe ability of the tumor to metastasize. In some examples, treatmentusing the methods disclosed herein prolongs the time of survival of thesubject (e.g., increases survival time by at least 6 months, at least 9months, at least 12 months, at least 2 years, at least 3 years, or evenat least 5 years relative to the absence of the therapy).

In particular examples, the method includes administering to the subjecta therapeutically effective amount of one or more agents that reducecellular invasion resulting from the interaction between OPN-5kD andCD44 receptor, thereby treating the tumor. In particular examples,cellular invasion is reduced by at least 10% (such as at least 20%, atleast 50%, or at least 90%), for example as compared to an amount ofcellular invasion in the absence of the therapeutic agent. Examples ofsuch agents include peptide fragments of OPN-5kD or antibodies specificto OPN-5kD (e.g., 5 kd106-13 D) that reduce the interaction betweenOPN-5kD and CD44 receptor and reduce cellular invasion. Further detailon the OPN-5kD antibodies that can be used therapeutically is providedbelow.

Particular examples of agents that can be used in the methods disclosedherein include therapeutic amounts of peptide fragments of OPN-5kD. Suchfragments include peptides of 5 to 50 amino acids (such as 5 to 25, 10to 44, 10 to 20, or 10 to 15 amino acids) that include at least 5contiguous amino acids of an OPN-5kD sequence (e.g., SEQ ID NO: 4), suchas at least 6, at least 7, at least 8, at least 8, at least 9, at least10 or at least 15 contiguous amino acids of SEQ ID NO: 4, for example5-20, 10-15, or 8-10 contiguous amino acids of SEQ ID NO: 4. Forexample, the therapeutic peptide can include or consist of 5, 6, 7, 8,9, 10, 12, 15, 20, or 30 contiguous amino acids of SEQ ID NO: 4. In oneexample, the peptide fragment of OPN-5kD include or consists of theamino acid sequence of SEQ ID NO: 5, 6, 7, or 8 or amino acids 30-36 ofSEQ ID NO: 4. One skilled in the art will appreciate that more than onepeptide fragment of OPN-5 kD can be used, such as one or more peptidesconsisting of SEQ ID NO: 5, 6, 7, or 8. In some examples the therapeuticpeptide is a fusion peptide, such as a fusion peptide that includes anyof SEQ ID NOS: 5-8.

Screening Subjects

Subjects that can benefit from the disclosed therapies include human andveterinary subjects. Subjects can be screened prior to initiating thedisclosed therapies, for example to determine whether the subject has atumor that overexpresses OPN, MMP-9, or both. The presence of a tumorthat overexpresses OPN, MMP-9, or both indicates that the tumor can betreated using the methods provided herein.

In one example, the tumor (or a portion thereof, such as a fine needleaspirate or other biopsy sample) is analyzed using immunodetectionmethods. For example, the biological sample can be incubated with anantibody that specifically binds to OPN or to MMP-9, or both antibodies.The primary antibody can include a detectable label. For example, theprimary antibody can be directly labeled, or the sample can besubsequently incubated with a secondary antibody that is labeled (forexample with a fluorescent label). The label can then be detected, forexample by microscopy, ELISA, flow cytometery, or spectrophotometry. Inanother example, the biological sample is analyzed by Western blottingfor the presence of OPN or to MMP-9. In one example, a subject isscreened by determining whether increased levels of the OPN-5kD fragmentis present in their serum (for example relative to a level present in aserum sample from a subject not having a tumor), for example using anantibody that specifically binds OPN-5kD (such as those described inExample 9).

As an alternative to analyzing the sample for the presence of proteins,the presence of nucleic acids can be determined. For example, thebiological sample can be incubated with primers that permit theamplification of OPN or to MMP-9, under conditions sufficient to permitamplification of OPN or to MMP-9. Exemplary methods include PCR andRT-PCR. In another example, the biological sample is incubated withprobes that can bind to OPN or to MMP-9 nucleic acid (such as cDNA,genomic DNA, or RNA (such as mRNA)) under high stringency conditions.The resulting hybridization can then be detected using methods known inthe art. In one example, a subject is screened by determining whetherincreased levels of OPN-c are present in the tumor (for example relativeto a level present in adjacent non-tumor cells from the same subject, orrelative to a level of OPN-a in the same tumor sample), for exampledetecting OPN-c (and in some examples also OPN-a) mRNA expression.

The presence of increased expression of OPN or MMP-9 indicates that thetumor overexpresses OPN or MMP-9 or both, and that the tumor is one thatcan be treated using the disclosed therapies. In one example, thepresence of increased OPN-5 kD in the serum, the presence of increasedOPN-c in the tumor, or both, indicates that the subject has a tumor thatcan be treated using the disclosed therapies.

Administration

Methods of administration of the disclosed therapeutic agents areroutine, and can be determined by a skilled clinician. For example, thedisclosed therapies (such as peptide fragments of OPN-5kD and antibodiesdirected to an epitope of OPN-5kD) can be administered via injection(for example via embolization), orally, topically, transdermally,parenterally, or via inhalation or spray. In a particular example, apeptide consisting of 5 to 20 contiguous amino acids of OPN-5kD or anantibody specific for OPN-5kD (such as 5 kd106-13 D) is administeredintravenously to a mammalian subject, such as a human.

The therapeutic compositions, such as those that include peptidefragments of OPN-5kD or antibodies specific for OPN-5kD, can furtherinclude one or more biologically active or inactive compounds (or both),such as anti-neoplastic agents and conventional non-toxicpharmaceutically acceptable carriers, respectively.

In a particular example, a therapeutic composition that includes atherapeutically effective amount of one or more agents that reducecellular invasion due to the interaction of OPN-5kD with CD44 (such aspeptide fragments of OPN-5kD or antibodies specific for OPN-5kD) furtherincludes one or more biologically inactive compounds. Examples of suchbiologically inactive compounds include, but are not limited to:carriers, thickeners, diluents, buffers, preservatives, and carriers.The pharmaceutically acceptable carriers useful for these formulationsare conventional (see Remington's Pharmaceutical Sciences, by E. W.Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995)). Ingeneral, the nature of the carrier will depend on the particular mode ofadministration being employed. For instance, parenteral formulations caninclude injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can include minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

In a particular example, a therapeutic composition that includes atherapeutically effective amount of one or more agents that reducecellular invasion due to the interaction of OPN-5kD with CD44 (such aspeptide fragments of OPN-5kD or antibodies specific for OPN-5kD) furtherincludes therapeutically effective amounts of one or more otherbiologically active compounds. Examples of biologically active compoundsinclude, but are not limited to: anti-neoplastic agents (such aschemotherapeutics), antibiotics, alkylating agents, antioxidants,adjuvants, and so forth (such as those listed below under “additionaltreatments”). However, one skilled in the art will appreciate thatpeptide fragments of OPN-5kD or antibodies specific for OPN-5 kD and theother biologically active compounds can also be administered separately(instead of in a single composition).

The therapeutically effective amount of the agents administered can varydepending upon the desired effects and the subject to be treated. In oneexample, the method includes daily administration of at least 1 μg ofone or more peptide fragments of OPN-5kD to the subject (such as a humansubject). In one example, a human is administered at least 1 μg ofpeptide daily or at least 1 mg peptide daily of one or more OPN-5kDfragments (such as one or more of SEQ ID NOS: 5-8), such as 10 μg to 100μg daily, 100 μg to 1000 μg daily, for example 10 μg daily, 100 μgdaily, or 1000 μg daily. For example the subject can receive at least 1μg (such as 1 μg to 100 μg, 5 μg to 50 μg, or 1 μg to 1000 μg)intravenously of each of one or more OPN-5kD fragments (such as one ormore of SEQ ID NOS: 5-8). In one example, the subject is administered atleast 1 mg intramuscularly (for example in an extremity) or topically ofeach of one or more OPN-5kD fragments (such as one or more of SEQ IDNOS: 5-8). For example, a human can be administered at least 1 μg dailyor at least 1 mg daily of one or more OPN-5kD fragments (such as one ormore of SEQ ID NOS: 5-8), such as 10 μg to 100 μg daily, 100 μg to 1000μg daily, for example 10 μg daily, 100 μg daily, or 1000 μg daily. Inone example, the subject is administered at least 1 μg (such as 1 μg to100 μg, 5 μg to 50 μg, or 1 μg to 1000 μg) intravenously of each of oneor more OPN-5kD fragments (such as one or more of SEQ ID NOS: 5-8). Inone example, the subject is administered at least 1 mg intramuscularly(for example in an extremity) or topically of each of one or moreOPN-5kD fragments (such as one or more of SEQ ID NOS: 5-8). The dosagecan be administered in divided doses (such as 2, 3, or 4 divided dosesper day), or in a single dosage daily.

Therapeutic amounts of OPN-5kD specific antibodies disclosed herein(e.g., 5 kd106-13 D or a humanized form thereof, a chimeric formthereof, or a fragment thereof) can also be administered. In someexamples, therapeutic amounts are amounts which eliminate or reduce thepatient's tumor burden, or which prevent or reduce the proliferation ofmetastatic cells. The dosage will depend on many parameters, includingthe nature of the tumor, patient history, patient condition, thepossible co-use of other oncolytic agents, and methods ofadministration. Methods of administration include injection (e.g.,parenteral, subcutaneous, intravenous, intraperitoneal, etc.) for whichthe antibodies are provided in a nontoxic pharmaceutically acceptablecarrier such as water, saline, Ringer's solution, dextrose solution, 5%human serum albumin, fixed oils, ethyl oleate, or liposomes. Typicaldosages may range from about 0.01 to about 20 mg/kg, such as from about0.1 to about 10 mg/kg. Other methods of administration include oral andtransdermal (such as at least 1 mg, for example 1-1000 mg). Acceptablecarriers for oral ingestion include pharmaceutically acceptable liquidcarriers or pharmaceutically acceptable solid carriers in the form oftablets, capsules, caplets, or gel-seals. Other effective methods ofadministration and dosages may be determined by routine experimentationand are within the scope of this invention.

Therapeutic methods employing OPN-5kD specific antibodies or peptidefragments of OPN-5kD can be combined with chemotherapy, surgery, andradiation therapy, depending on type of the tumor, patient condition,other health issues, and a variety of factors. The methods can alsoinclude immunoconjugates for targeted immunotoxin-mediated therapy,wherein OPN-5kD antibodies are covalently or non-covalently conjugatedto various cytotoxic agents, further enhancing toxicity to targetedcells. See, for example, U.S. Pat. No. 5,872,223. Such agents, includingvarious bacterial toxins (e.g., Pseudomonas exotoxin), ricin A-chain,daunorubicin, methotrexate, and ribosome inhibitors (e.g.,trichosantin). Also, OPN-5kD antibodies can be labeled with alpha, beta,or Auger electron emitters, resulting in immunoconjugates for targetedradiotherapy.

Thus, OPN-5kD specific antibodies can be used in a variety of methodsand compositions for detecting and treating metastatic disease.

In particular examples, the subject is administered the therapeuticcomposition that includes OPN-5kD specific antibodies, or one or morepeptide fragments of OPN-5 kD, on a multiple daily dosing schedule, suchas at least two consecutive days, 10 consecutive days, and so forth, forexample for a period of weeks, months, or years. In one example, thesubject is administered the therapeutic composition for a period of atleast 30 days, such as at least 2 months, at least 4 months, at least 6months, at least 12 months, at least 24 months, or at least 36 months.

Additional Treatments

In particular examples, prior to, during, or following administration ofa therapeutic amount of an agent that reduces cellular invasion due tothe interaction of OPN-5kD with CD44 (such as one or more peptidefragments of OPN-5kD or OPN-5 kD antibodies), the subject can receiveone or more other therapies. In one example, the subject receives one ormore treatments to remove or reduce the tumor prior to administration ofa therapeutic amount of one or more agents that reduce cellular invasiondue to the interaction of OPN-5kD with CD44.

Examples of such therapies include, but are not limited to, surgicaltreatment for removal or reduction of the tumor (such as surgicalresection, cryotherapy, or chemoembolization), as well as anti-tumorpharmaceutical treatments which can include radiotherapeutic agents,anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agentsand antioxidants, kinase inhibitors, and other agents. In one example,at least part of the tumor is surgically or otherwise excised or reducedin size or volume prior to administering the therapeutically effectiveamount of the antibody or peptide. Particular examples of additionaltherapeutic agents can that can be used include microtubule bindingagents, DNA intercalators or cross-linkers, DNA synthesis inhibitors,DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzymeinhibitors, gene regulators, and angiogenesis inhibitors. These agents(which are administered at a therapeutically effective amount) andtreatments can be used alone or in combination. Methods and therapeuticdosages of such agents are known to those skilled in the art, and can bedetermined by a skilled clinician.

“Microtubule binding agent” refers to an agent that interacts withtubulin to stabilize or destabilize microtubule formation therebyinhibiting cell division. Examples of microtubule binding agents thatcan be used in conjunction with the disclosed therapy include, withoutlimitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine(navelbine), the epothilones, colchicine, dolastatin 15, nocodazole,podophyllotoxin and rhizoxin. Analogs and derivatives of such compoundsalso can be used and are known to those of ordinary skill in the art.For example, suitable epothilones and epothilone analogs are describedin International Publication No. WO 2004/018478. Taxoids, such aspaclitaxel and docetaxel, as well as the analogs of paclitaxel taught byU.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.

Suitable DNA and/or RNA transcription regulators, including, withoutlimitation, actinomycin D, daunorubicin, doxorubicin and derivatives andanalogs thereof also are suitable for use in combination with thedisclosed therapies.

DNA intercalators and cross-linking agents that can be administered to asubject include, without limitation, cisplatin, carboplatin,oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil,cyclophosphamide and derivatives and analogs thereof.

DNA synthesis inhibitors suitable for use as therapeutic agents include,without limitation, methotrexate, 5-fluoro-5′-deoxyuridine,5-fluorouracil and analogs thereof.

Examples of suitable enzyme inhibitors include, without limitation,camptothecin, etoposide, formestane, trichostatin and derivatives andanalogs thereof.

Suitable compounds that affect gene regulation include agents thatresult in increased or decreased expression of one or more genes, suchas raloxifene, 5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen,4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.

“Angiogenesis inhibitors” include molecules, such as proteins, enzymes,polysaccharides, oligonucleotides, DNA, RNA, and recombinant vectors,and small molecules that function to reduce or even inhibit blood vesselgrowth. Angiogenesis is implicated in most types of human solid tumors.Angiogenesis inhibitors are known in the art and examples of suitableangiogenesis inhibitors include, without limitation, angiostatin K1-3,staurosporine, genistein, fumagillin, medroxyprogesterone, suramin,interferon-alpha, metalloproteinase inhibitors, platelet factor 4,somatostatin, thromobospondin, endostatin, thalidomide, and derivativesand analogs thereof.

Exemplary kinase inhibitors include Gleevac, Iressa, and Tarceva thatprevent phosphorylation and activation of growth factors.

Antibodies that can be used include Herceptin and Avastin that blockgrowth factors and the angiogenic pathway.

Other therapeutic agents, for example anti-tumor agents, that may or maynot fall under one or more of the classifications above, also aresuitable for administration in combination with the disclosed therapies(such as those that include one or more peptide fragments of OPN-5kD oran OPN-5kD specific antibody). By way of example, such agents includeadriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivativesand analogs thereof.

In one example, the therapeutic peptide composition (such as OPN-5kD orpeptide fragments thereof) or is injected into the subject in thepresence of an adjuvant, thus generating an immune response to OPN-5kDwherein such immune response decreases OPN-5kD binding to CD44. Anadjuvant is an agent that when used in combination with an immunogenicagent augments or otherwise alters or modifies a resultant immuneresponse. In some examples, an adjuvant increases the titer ofantibodies induced in a subject by the immunogenic agent. In oneexample, the one or more peptides are administered to the subject as anemulsion with IFA and sterile water for injection (for example anintravenous or intramuscular injection). Incomplete Freund's Adjuvant(Seppic, Inc.) can be used as the Freund's Incomplete Adjuvant (IFA)(Fairfield, N.J.). In some examples, IFA is provided in 3 ml of amineral oil solution based on mannide oleate (Montanide ISA-51). At thetime of injection, the peptide(s) is mixed with the Montanide ISA.51 andthen administered to the subject. Other adjuvants can be used, forexample, Freund's complete adjuvant, B30-MDP, LA-15-PH, montanide,saponin, aluminum hydroxide, alum, lipids, keyhole lympet protein,hemocyanin, a mycobacterial antigen, and combinations thereof.

In some examples, the subject receiving a therapeutic composition (suchas one or more peptide fragments of OPN-5kD or OPN-5kD antibodies) isalso administered interleukin-2 (IL-2), for example via intravenousadministration. In particular examples, IL-2 (Chiron Corp., Emeryville,Calif.) is administered at a dose of at least 500,000 IU/kg as anintravenous bolus over a 15 minute period every eight hours beginning onthe day after administration of the peptides and continuing for up to 5days. Doses can be skipped depending on subject tolerance.

In some examples, the therapeutic compositions can be co-administeredwith a fully human antibody to cytotoxic T-lymphocyte antigen-4(anti-CTLA-4). In some examples subjects receive at least 1 mg/kganti-CTLA-4 (such as 3 mg/kg every 3 weeks or 3 mg/kg as the initialdose with subsequent doses reduced to 1 mg/kg every 3 weeks).

In one example, at least a portion of the tumor (such as a metastaticHCC) is surgically removed (for example via cryotherapy), irradiated,chemically treated (for example via chemoembolization) or combinationsthereof, prior to administration of the disclosed therapies. Forexample, a subject having HCC can have all or part of the tumorsurgically excised prior to administration of the disclosed therapies.In another particular example, the subject has HCC and is administeredradiation therapy, chemoembolization therapy, or both, prior toadministration of the disclosed therapies.

Therapeutic Compositions

Another aspect of the disclosure includes pharmaceutical compositionsprepared for administration to a subject and which include atherapeutically effective amount of one or more of the currentlydisclosed compounds. Such compositions can also be part of a kit. Forexample, compositions that include a therapeutic amount of an agent thatdecreases or inhibits cellular invasion that results from theinteraction of OPN-5kD with CD44 receptor can be formulated for use intreating a tumor that overexpresses OPN, such as HCC. Particularexamples of such agents include OPN-5kD specific antibodies and peptidefragments of OPN-5kD, such as the peptide sequences shown in SEQ ID NOS:5-8.

In one example, compositions include a therapeutic amount of one or moreagents that decrease or inhibit tumor cellular invasion (for example areduction of at least 10%, at least 20%, at least 50%, or even at least90%) such as by decreasing the binding of OPN-5kD to the CD44 receptor.Such compositions can include one or more additional biologically activeagents, one or more biologically inactive compounds, or combinationsthereof. Examples of such biologically active agents are describedabove, and can include anti-neoplastic agents. Examples of biologicallyinactive agents are described above, and can include pharmaceuticallyacceptable carriers. In a specific example, the therapeutic compositionincludes an antibody to cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4).

Particular examples of agents that can be used in the disclosedcompositions include peptide fragments of OPN-5kD that decrease tumorcellular invasion resulting from the interaction of OPN-5kD with CD44receptor. One example of OPN-5kD peptide fragments includes peptides of5 to 60 amino acids that have at least 5 contiguous amino acids ofOPN-5kD (such as at least 5 contiguous amino acids of SEQ ID NO: 4),such as at least 6, at least 7, at least 8, at least 9, at least 10, atleast 12, at least 15, or even at least 20 contiguous amino acids ofOPN-5kD, for example 5-30, 5-25, 6-20, 6-12, or 8-10 contiguous aminoacids of OPN-5kD (such as this number of contiguous amino acids of SEQID NO: 4). Exemplary examples of such peptides are shown in SEQ ID NOS:5-8.

In a particular example, the composition includes at least two peptidefragments of OPN-5kD. For example, the composition can include a mixtureof at least 2 of the peptides shown in SEQ ID NOS: 5-8, such as acomposition that includes 2, 3, or 4 of these peptides. Exemplarycombinations include the peptides shown in SEQ ID NOS: 5 and 6, SEQ IDNOS: 5 and 7, SEQ ID NOS: 5 and 8, SEQ ID NOS: 6 and 7, SEQ ID NOS: 6and 8, SEQ ID NOS: 7 and 8, SEQ ID NOS: 5, 6, and 7, SEQ ID NOS: 5, 6,and 8, SEQ ID NOS: 5, 7, and 8, SEQ ID NOS: 6, 7, and 8, or SEQ ID NOS:5, 6, 7, and 8. In one example, the composition includes one or morepeptides consisting of the amino acid sequence shown in SEQ ID NO: 5, 6,7, or 8.

In particular examples, the composition includes 1-1000 μg of one ormore peptide fragments of OPN-5kD that decrease tumor cellular invasionresulting from the interaction of OPN-5kD with CD44 receptor, such as1-1000 μg of one or more of SEQ ID NOS: 5-8. In some examples, thecomposition includes 1-1000 mg of one or more peptide fragments ofOPN-5kD that decrease tumor cellular invasion resulting from theinteraction of OPN-5kD with CD44 receptor, such as 1-1000 mg of one ormore of SEQ ID NOS: 5-8.

In another particular example, the composition includes one or moreOPN-5kD specific antibodies, such as 5 kd106-13 D (or other monoclonalantibody or humanized form thereof that recognizes the epitope shown inamino acids 30-36 of SEQ ID NO: 4). Such antibodies bind to OPN-5kD orfragments thereof and inhibit or decrease binding to CD44 receptors. Inparticular examples, the composition includes 1-1000 μg or 1-1000 mg ofone or more OPN-5kD specific antibodies that decrease tumor cellularinvasion resulting from the interaction of OPN-5kD with CD44 receptor.Details on such antibodies are provided below.

Methods of Diagnosing and Prognosing a Tumor

Metastasis is a major complication in the pathogenesis of tumors, suchas HCC, and is typically indicative of poor prognosis. It is shownherein that OPN-5kD abundance in HCC cell lines correlates with degreeof metastatic potential. Without wishing to be bound to a particulartheory, it is proposed that the interaction of OPN and MMP-9 is relatedto enhanced HCC tumor cell metastasis. The abundance of the OPN-5kDfragment in HCC cells correlates to degree of metastatic potential. Itis also shown herein that OPN-c mRNA expression correlates withmetastatic HCC but not non-metastatic HCC in non-cancerous tissueadjacent to primary HCC lesions. Based on these observations, methods ofdiagnosing or prognosing a tumor that overexpresses OPN, MMP-9, or both,based on detecting OPN-5kD, OPN-c, or both, are disclosed. In someexamples, such methods can be used to identify those subjects that willbenefit from the disclosed treatment methods. For example, suchdiagnostic methods can be performed prior to the subject undergoing thetreatments described herein.

In some examples the method is performed in vitro, for example when thebiological sample is removed from the subject. In some examples themethod is carried out in vivo for example by administering the disclosedantibodies, humanized form thereof or fragment thereof to a subject,such as a subject that has or is suspected of having an OPN-expressingtumor, such as HCC.

In one example, detection of the OPN-5kD fragment (such as SEQ ID NO: 4or a variant thereof, such as a polymorphism thereof) in a biologicalsample from the subject is used to diagnose or prognose a tumor thatoverexpresses OPN, MMP-9, or both (such as HCC). Methods of detecting apeptide in a sample, such as OPN-5kD, are known in the art and areroutine. In some examples, the relative amount of OPN-5kD present isdetermined, for example by quantitating the amount of OPN-5kD present.For example, the relative or absolute quantity of OPN-5kD in a samplecan be determined.

In another example, detection of OPN-c mRNA (such as an mRNAcorresponding to SEQ ID NO: 3 or 9, or a variant thereof, such as apolymorphism thereof) in a biological sample from the subject is used todiagnose or prognose a tumor that overexpresses OPN, MMP-9, or both(such as metastatic HCC). Methods of detecting a nucleic acid moleculein a sample, such as OPN-c, are known in the art and are routine. Insome examples, the relative amount of OPN-c mRNA present is determined,for example by quantitating the amount of OPN-c mRNA present. Forexample, the relative or absolute quantity of OPN-c mRNA in a sample canbe determined. However, one skilled in the art will recognize that OPN-cprotein can be detected as an alternative to detecting OPN-c nucleicacid molecules. In some examples, OPN-c expression is measured in atumor sample and an adjacent non-tumor sample, wherein an increase inOPN-c expression (such as an increase of at least 2-fold, at least3-fold, or at least 5-fold) in the tumor sample relative to OPN-cexpression in the non-tumor sample indicates that the subject has atumor with increased metastatic potential. In other examples, OPN-c andOPN-a expression are measured in the tumor sample, wherein an increasein OPN-c expression (such as an increase of at least 2-fold, at least3-fold, or at least 5-fold) relative to OPN-a expression in the tumorsample indicates that the subject has an OPN-overexpressing tumor.

In some examples, detection of both OPN-5kD protein and OPN-c mRNA (anin some examples also OPN-a mRNA) in one or more biological samplesobtained from the subject (such as serum or a tumor sample or adjacentnon-tumor sample) are used to diagnose or prognose a tumor thatoverexpresses OPN, MMP-9, or both (such as metastatic HCC).

Biological Samples

A biological sample is typically obtained from a mammal, such as a rat,mouse, cow, dog, guinea pig, rabbit, or primate, such as a human. Thusis some examples a subject, such as a human subject, is selected and abiological sample from that subject is tested for the presence of anOPN-expressing tumor (for example by detecting OPN-5 kD, OPN-c, and/orOPN-a). Methods of obtaining a biological sample from a subject areknown in the art. For example, methods of obtaining a blood sample or afraction thereof (such as serum) are routine. Similarly, a sample from atumor (or adjacent non-tumor tissue) that contains cellular material canbe obtained by surgical excision of all or part of the tumor, bycollecting a fine needle aspirate from the tumor, as well as othermethods known in the art. If desired, the sample can be concentrated orpurified before use. For example, proteins or nucleic acids can beisolated from the sample. In some examples, the sample is filteredbefore use, for example to remove undesired proteins (e.g.,fractionating serum to remove full-length OPN while retaining OPN-5kD).Alternatively, the sample can be used directly. In particular examples,a serum sample obtained from the subject is analyzed to determine if itcontains detectable levels of OPN-5kD. In particular examples, a tumorsample obtained from the subject is analyzed to determine if it containsdetectable levels of OPN-c (and in some examples also OPN-a) mRNA orprotein.

Detection of OPN-5kD Peptide In Vitro

In particular examples, a sample obtained from the subject is analyzedto determine if it contains detectable levels of the OPN-5kD fragment,such as a serum sample.

Methods of detecting proteins are routine. In some examples,immunoassays are used to detect the presence of the OPN-5kD proteinfragment in the sample. Generally, immunoassays include the use of oneor more specific binding agents (such as antibodies) that cansubstantially only bind to OPN-5kD. Such binding agents can include adetectable label (such as a radiolabel, fluorophore or enzyme), thatpermits detection of the binding to the protein. Exemplary immunoassaysthat can be used include, but are not limited to: Western blotting,ELISA, fluorescence microscopy, and flow cytometry.

In one example, the specific binding agent is an antibody, such as apolyclonal or monoclonal antibody, or fragment thereof. In someexamples, the antibody is a humanized antibody. In some examples, theantibody is a chimeric antibody. If desired, the antibody can include adetectable label to permit detection and in some cases quantitation ofthe OPN-5kD/antibody complex.

In a particular example, the antibody used is 5 kd106-13 D. For example,the method can include contacting the biological sample (e.g., serum)with the monoclonal 5 kd106-13 D antibody, a chimeric form thereof, ahumanized form thereof, or a functional fragment thereof underconditions in which an immune complex will form between the 5 kd106-13 Dantibody, chimeric form thereof, humanized form thereof, or functionalfragment thereof and the OPN-5kD fragment. In some examples, thepresence (or absence) of the immune complex is then detected. Thepresence of the immune complex indicates the presence of the OPN-5kDfragment, and thus an OPN-expressing or -overexpressing tumor such asHCC. In some embodiments a second antibody (such as an antibody thatrecognizes a mouse IgG) (which can be detectably labeled) thatspecifically binds the 5 kd106-13 D antibody, a chimeric form thereof,or a humanized form thereof or a functional fragment thereof is used todetect OPN-5kD.

The presence of detectable signal above background or control levelsindicates that the presence of OPN-5kD peptide in the sample. Forexample, the level of OPN-5 kD detected can be compared to a control orreference value, such as a value that represents a level of OPN-5kDexpected if a tumor that overexpresses OPN is present in the subject(such as metastatic HCC), a level of OPN-5kD expected if a tumor thatoverexpresses OPN is absent in the subject (such as HCC or a normalsubject), a level of OPN-5kD expected if a tumor that overexpresses OPNis has metastasized in the subject (such as metastatic HCC), orcombinations thereof.

In some examples, full-length OPN is also measured in the sample, andthe expression levels of OPN and OPN-5kD compared, for example to ratiosexpected for healthy individuals, subjects with a metastaticOPN-overexpressing tumor, or subjects with an non-metasticOPN-expressing tumor, wherein the tested ratio is compared to thecontrol ratio, and the control ratio that is most similar to the testratio indicates the diagnosis of the subject.

In some examples, detection of OPN-5kD at a level of at least twice(such as at least three or at least five times) that observed in asubject not having a tumor, indicates that that subject has a tumor thatoverexpresses OPN, has a tumor with metastatic potential, has a tumorthat has metastasized, has a poor prognosis, or combinations thereof.For example, if serum levels of OPN-5kD from a subject are twice thatobserved for a subject not having a tumor, such as an increase of atleast 100%, at least 200%, or at least 500%, this indicates that thesubject has a tumor that overexpresses OPN, has a tumor with metastaticpotential, has a tumor that has metastasized, has a poor prognosis, orcombinations thereof.

For example, if the level of OPN-5kD detected in the subject's sample issignificantly less than the level of OPN-5kD expected if a tumor thatoverexpresses OPN is present in the subject, is similar to or less thanthe level of OPN-5kD expected if a tumor that overexpresses OPN is notpresent in the subject, or both, this indicates that the subject doesnot have a tumor that overexpresses OPN. In another example, if thelevel of OPN-5kD detected in the subject's sample is similar or greaterthan the level of OPN-5kD expected if a tumor that overexpresses OPN ispresent in the subject, is significantly greater than the level ofOPN-5kD expected if a tumor that overexpresses OPN is not present in thesubject, or both, this indicates that the subject has a tumor thatoverexpresses OPN, such as HCC. In another example, if the level ofOPN-5kD detected in the subject's sample is similar or greater than thelevel of OPN-5kD expected if a tumor that overexpresses OPN is presentin the subject, is significantly greater than the level of OPN-5kDexpected if a tumor that overexpresses OPN is not present in thesubject, is similar to or greater than the level of a level of OPN-5kDexpected if a tumor that overexpresses OPN is has metastasized in thesubject, or combinations thereof, this indicates that the subject has ametastatic tumor that overexpresses OPN, such as metastatic HCC.

Detection of OPN-5kD In Vivo

OPN-5kD specific antibodies can also be used in vivo for diagnosis. Forexample, the 5 kd106-13 D antibody, a chimeric form thereof, or ahumanized form thereof or a fragment thereof disclosed herein can alsobe used to detect OPN-expressing or over-expressing tumors (e.g., HCC),in vivo. The antibodies disclosed herein can also be used to detect HCCand metastatic HCC in vivo. In one embodiment, a 5 kd106-13 D antibody,a chimeric form thereof, or a humanized form thereof or a fragmentthereof is administered to the subject for a sufficient amount of timefor the antibody to localize to the tumor in the subject and to form animmune complex with the tumor. The immune complex can then be detectedfor example radiolocalization, radioimaging, MRI, PET scan, orfluorescence imaging, for example by using a detectibly labeledantibody, humanized form thereof or functional fragment thereof. Oncedetected, in an ectopic location (as in a tumor) the test results can beused to assist in or guide surgical or other excision of a tumor.

In vivo imaging methods can also be utilized with the OPN-5kD antibodiesdisclosed herein. These technologies include magnetic resonance imaging(for example using a biotinylated antibody and avidin-iron oxide),positron emission tomography (for example using an ¹¹¹indium-labeledmonoclonal antibody), and optical imaging (for example using luciferaseor green fluorescent protein labeled antibodies). In one example,magnetic resonance imaging is utilized. In the setting of magneticresonance imaging, contrast agent detection can be greatly impacted bymagnetic resonance scanner field strength. Increased field strengthsprovide improvements by orders of magnitude in the ability to detectcontrast agents (Hu et al., Annu Rev Biomed Eng. 6:157-184, 2004;Wedeking et al., Magn. Reson. Imaging. 17:569-575, 1999). For example,the limit of detection of gadolinium at 2 tesla (T) is ˜30 μM. At 4 Tthe limit of detection is reduced to ˜1 μM. With newly available 7 to 12T scanners one would expect to detect low (10-100) nM concentrations ofthis contrast agent. Similar sensitivity can also be identified usingcontrast agents such as iron oxide.

Detection of OPN-c and OPN-a

In particular examples, a sample obtained from the subject is analyzedto determine if it contains detectable levels of OPN-c and in someexamples also OPN-a, such as a tumor sample, a sample adjacent to thetumor, or both. In particular examples, OPN-a and OPN-c nucleic acidmolecules (such as mRNA) are measured. However, one skilled in the artwill appreciate that OPN-c protein could also be measured, for exampleusing routine immunoassays known in the art (such as those describedabove).

Methods of detecting nucleic acid molecules are routine. In particularexamples, a tumor sample and a sample adjacent to the tumor obtainedfrom the subject are analyzed to determine if each contains detectablelevels of OPN-c nucleic acid molecules, such as cDNA or mRNA. Forexample, assays that permit detection of nucleic acids can be used.Exemplary assays that can be used include, but are not limited to:Northern blotting, Southern blotting, PCR (such as RT-PCR), and DNAarrays. For example, OPN-c can be amplified from a sample using PCR, andthe amplicons detected and in some examples quantitated, wherein thepresence of detectable amplicons above background or control levelsindicates that the tumor (or adjacent tissue) expresses OPN-c nucleicacid molecules. In one example, a nucleic acid probe that hybridizes toan OPN-c nucleic acid is contacted with the sample. For example, theprobe can be incubated with the sample under high stringency conditions(such as when the hybridization is performed at about 42° C. in ahybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC,5×Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA,50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5×10⁷cpm/μm), while the washes are performed at about 65° C. with a washsolution containing 0.2×SSC and 0.1% sodium dodecyl sulfate), whereinthe presence of detectable signal from the probe above background orcontrol levels indicates that the tumor (or adjacent tissue) expressesOPN-c nucleic acid molecules.

The presence of detectable signal above background or control levelsindicates that the presence of OPN-c nucleic acid molecules in thesample. For example, the level of OPN-c detected can be compared to acontrol or reference value, such as a value that represents a level ofOPN-c expected if a tumor is or is not metastatic (such as metastaticHCC). In one example, the level of OPN-c detected in a tumor sample iscompared to the level of OPN-c detected in an adjacent non-tumor sample.

In some examples, detection of at least twice (such as at least 3-timesor at least 5-times) the relative amount of OPN-c mRNA observed in atumor sample, as compared to the relative amount of OPN-c mRNA in anadjacent non-tumor sample from the same subject, indicates that thatsubject has a tumor with metastatic potential, has a tumor that hasmetastasized, has a poor prognosis, or combinations thereof. Forexample, if tumor levels of OPN-c mRNA are twice that observed for anadjacent non-tumor sample, such as an increase of at least 100%, atleast 200%, or at least 500%, this indicates that the subject has atumor with metastatic potential, has a tumor that has metastasized, hasa poor prognosis, or combinations thereof.

In some examples, detection of statistically similar relative amounts ofOPN-c mRNA observed in a tumor sample, as compared to the relativeamount of OPN-c mRNA in an adjacent non-tumor sample from the samesubject, indicates that that subject does not have a tumor withmetastatic potential, does not have a tumor that has metastasized, has agood prognosis, or combinations thereof.

In some examples, the ratio of OPN-a to OPN-c in tumor cells isdetermined. It is shown herein that the ratio for OPN-a versus OPN-c inmetastatic tumors is slightly lower than for non-metastatic tumors,indicating higher levels of OPN-c in metastasis (for example a ratio of2.27 versus 2.85). Thus methods are provided for measuring OPN-a andOPN-c expression in a tumor sample. OPN-a can be detected using methodssimilar to that described above for OPN-c. Expression levels of OPN-cexpression and OPN-a expression in the tumor sample are compared,wherein a statistically significant increase in OPN-c expression (suchas an increase of at least 20%, at least 50%, or at least 80%) in thetumor sample compared to the amount of OPN-a expression in the tumorsample indicates that the subject has an OPN-expressing tumor.

Monitoring Response to Therapy

The disclosed OPN-5kD antibodies and fragments thereof can be used tomonitor response to therapy. The number and or mass of OPN-expressingtumor cells (e.g., HCC cells), such as the cells present in a subject,can be determined using the methods disclosed herein. In one embodiment,an increase in the number or mass of OPN-expressing tumor cells (e.g.,HCC cells), as compared to a control, such as the number or mass ofOPN-expressing tumor cells (e.g., HCC cells) at an earlier time point,indicates that the tumor is progressing and that the therapy as noteffective in reducing tumor burden. Conversely, a decrease in the numberor mass of OPN-expressing tumor cells (e.g., HCC cells), as compared toa control, such as the number or mass of OPN-expressing tumor cells(e.g., HCC cells) at an earlier time point, indicates that the tumor isregressing and that the therapy is effective. A control can be astandard value, or the number or mass of OPN-expressing tumor cells(e.g., HCC cells) in a sample from a subject not afflicted with a tumoror the number or mass of OPN-expressing tumor cells (e.g., HCC cells) ina sample from the subject at an earlier time point, for example prior totherapy.

Kits

Provided by this disclosure are kits that can be used to diagnose,prognose, or treat a tumor that overexpresses OPN (or combinationsthereof). The disclosed kits can include instructional materialsdisclosing means of use of the compositions in the kit. Theinstructional materials can be written, in an electronic form (such as acomputer diskette or compact disk) or can be visual (such as videofiles).

Kits are provided that can be used in the therapies and diagnosticassays disclosed herein. For example, kits can include one or more ofthe disclosed therapeutic compositions (such as a composition includingone or more peptide fragments of OPN-5 kD or OPN-5kD specificantibodies), one or more of the disclosed diagnostic compositions (suchas an antibody that specifically binds OPN-5kD), or combinationsthereof. Such agents can be present in separate vials.

One skilled in the art will appreciate that the kits can include otheragents to facilitate the particular application for which the kit isdesigned. Thus, for example, the kit may additionally contain means ofdetecting a label (such as enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a secondary antibody, or the like). The kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

In one example, a kit is provided that includes reagents for detectingOPN-5kD in a biological sample, such as serum. Kits for detectingOPN-5kD can include one or more antibodies that specifically bindOPN-5kD, such as any of the antibodies disclosed herein. For example, anOPN-5kD-specific antibody disclosed herein (e.g., 5 kd106-13 D), achimeric form thereof, a humanized form thereof, or a fragment thereofcan be part of the disclosed kits. In some embodiments, an OPN-5kDantibody fragment, such as an Fv fragment is included in the kit. In oneexample, such as for in vivo uses, the antibody can be a scFv fragment.In a further embodiment, the antibody is labeled (for example, with afluorescent, radioactive, or an enzymatic label). Such a diagnostic kitcan additionally contain means of detecting a label (such as enzymesubstrates for enzymatic labels, filter sets to detect fluorescentlabels, appropriate secondary labels such as a secondary antibody, orthe like), as well as buffers and other reagents routinely used for thepractice of a particular diagnostic method.

In one example, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting the OPN-5kD fragment in a biologicalsample generally includes the steps of contacting the sample with anantibody which specifically reacts with OPN-5kD (e.g., 5 kd106-13 D)under immunologically reactive conditions. The antibody is allowed tospecifically bind under immunologically reactive conditions to form animmune complex, and the presence of the immune complex (bound antibody)is detected directly or indirectly.

In one example, a kit is provided for treating a tumor thatoverexpresses OPN, such as HCC. For example, such kits can include oneor more of the disclosed therapeutic compositions (such as a compositionincluding an OPN-5kD specific antibody or including one or more peptidefragments of OPN-5kD). Such a diagnostic kit can additionally containother therapeutic molecules, such as therapeutic doses of IL-2,anti-CTLA-4, an adjuvant, or combinations thereof.

OPN-5kD Antibodies

The present disclosure provides antibodies that specifically bindOPN-5kD, such as bind to the epitope EELNGAY (amino acids 30-36 of SEQID NO: 4). One exemplary anti-OPN-5kD monoclonal antibody is 5 kD106-13D. The disclosed OPN-5 kd antibodies are useful for both therapeutic anddiagnostic purposes. In some examples OPN-5kD specific antibodies havean equilibrium constant (K_(d)) of 1 nM or less. For example, antibodiesare provided that bind human OPN-5kD with a binding affinity of 0.1×10⁻⁸M, at least about 0.3×10⁻⁸M, at least about 0.5×10⁻⁸M, at least about0.75×10⁻⁸ M, at least about 1.0×10⁻⁸M, at least about 1.3×10⁻⁸ Mat leastabout 1.5×10⁻⁸M, or at least about 2.0×10⁻⁸ M. In additional examples,the antibody (such as a human monoclonal) binds an epitope of OPN-5kDwith an equilibrium disassociation constant (K_(d)) of 1 nM or less. Inparticular examples, the antibody can be used to diagnose a tumor, or beused to predict the metastatic potential of the tumor. In otherexamples, the antibodies can be used in therapeutic compositions totreat a subject. The disclosed antibodies can be part of a kit, such asa kit used to diagnose a tumor, such as HCC (such as metastatic HCC).

In one example, the antibody is a monoclonal antibody. The monoclonalantibody can be of any isotype, such as an IgM or an IgG antibody, forexample IgG₁ or an IgG₂. The class of an antibody that specificallybinds OPN-5kD can be switched with another. In one aspect, a nucleicacid molecule encoding V_(L) or V_(H) is isolated using methodswell-known in the art, such that it does not include any nucleic acidsequences encoding the constant region of the light or heavy chain,respectively. The nucleic acid molecule encoding V_(L) or V_(H) is thenoperatively linked to a nucleic acid sequence encoding a C_(L) or C_(H)from a different class of immunoglobulin molecule. This can be achievedusing a vector or nucleic acid molecule that includes a C_(L) or C_(H)chain, as known in the art. For example, an antibody that specificallybinds OPN-5kD that was originally IgM can be class switched to an IgG.Class switching can be used to convert one IgG subclass to another, suchas from IgG₁ to IgG₂.

Isolated monoclonal antibodies specific for the OPN-5kD fragment areprovided that are produced by the hybridoma 5 kd106-13 D, as arehumanized forms of the 5 kd106-13 D antibody, chimeras of the 5 kd106-13D antibody, and functional fragments of 5 kd106-13 D. The 5 kd106-13 Dantibody specifically binds the OPN-5kD fragment produced by HCC cells.Generally, the monoclonal antibodies produced by the 5 kd106-13 Dhybridoma include a variable heavy (V_(H)) and a variable light (V_(L))chain and specifically bind the OPN-5kD fragment. For example, the 5kd106-13 D antibody can specifically bind the OPN-5kD fragment, such asan epitope thereof (e.g., amino acids 30-36 of SEQ ID NO: 4) with anaffinity constant of at least 10⁶ M⁻¹, such as at least 10⁷ M⁻¹, atleast 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, or at least 10⁹ M⁻¹. Hybridoma cellsand their progeny that secrete the monoclonal antibody 5 kd106-13 D arealso encompassed by this disclosure.

Antibody fragments are encompassed by the present disclosure, such asFab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and are capable of binding the epitopic determinant onOPN-5kD (e.g., EELNGAY, amino acids 30-36 of SEQ ID NO: 4). Theseantibody fragments retain the ability to selectively bind with theantigen. Methods of making these fragments are known in the art (see forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988). In a specific example, the antibodyis an Fv antibody. In other specific examples, the functional fragmentof the monoclonal antibody (e.g., 5 kd106-13 D) is a scFV fragment, anscFV₂ fragment, an Fv fragment, an Fab fragment, or an F(ab′)₂ fragment.

The production of chimeric antibodies, which include a framework regionfrom one antibody and the CDRs or SDRs from a different antibody, iswell known in the art. Thus chimeric and humanized forms of anti-OPN-5kDantibodies (e.g., the 5 kd106-13 D antibody) are provided herein. Theseantibodies include the CDRs (or SDRs) of an antibody specific for anepitope of the OPN-5kD fragment (e.g., the 5 kd106-13 D antibody) andframework regions from a different antibody. In one example, theframework regions are human. In some embodiments, a humanized antibodythat specifically binds the OPN-5kD fragment is a humanized form of the5 kd106-13 D monoclonal antibody (or other antibody specific for anepitope of the OPN-5kD fragment) or a functional fragment thereof. Inone example the sequence of the specificity determining regions (SDRs)of each CDR from the 5 kd106-13 D monoclonal antibody (or other antibodyspecific for an epitope of the OPN-5kD fragment) is determined. Residuesoutside the SDRs (non-ligand contacting sites) can be substituted andthe monoclonal antibody retains its ability to bind the OPN-5kDfragment. The antibody or antibody fragment can be a humanizedimmunoglobulin having complementarity determining regions (CDRs) fromthe 5 kd106-13 D monoclonal antibody (or other antibody specific for anepitope of the OPN-5kD fragment) produced by the above-describedhybridoma and immunoglobulin and heavy and light chain variable regionframeworks from human acceptor immunoglobulin heavy and light chainframeworks. Generally, the humanized immunoglobulin specifically bindsto the OPN-5kD fragment or an epitope thereof (such as EELNGAY, aminoacids 30-36 of SEQ ID NO: 4) with an affinity constant of at least 10⁷M⁻¹, such as at least 10⁸ M⁻¹ at least 5×10⁸ M⁻¹ or at least 10⁹ M⁻¹.

Humanized monoclonal antibodies can be produced by transferring donorCDRs from heavy and light variable chains of the donor mouseimmunoglobulin (such as the 5 kd106-13 D monoclonal antibody) into ahuman variable domain, and then substituting human residues in theframework regions when required to retain affinity. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of the constantregions of the donor antibody. Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyenet al., Science 239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci.U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; andSinger et al., J. Immunol. 150:2844, 1993. The antibody may be of anyisotype, but in several embodiments the antibody is an IgG, includingbut not limited to, IgG₁, IgG₂, IgG₃ and IgG₄.

In one embodiment, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 65% identical tothe sequence of the donor immunoglobulin heavy chain variable regionframework. Thus, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 75%, at leastabout 85%, at least about 95%, or at least about 99% identical to thesequence of the donor immunoglobulin heavy chain variable regionframework. The sequences of the heavy and light chain frameworks areknown in the art. Human framework regions, and mutations that can bemade in a humanized antibody framework regions, are known in the art(see, for example, in U.S. Pat. No. 5,585,089).

Exemplary human antibodies LEN and 21/28 CL are of use in providingframework regions. Exemplary light chain frameworks of human MAb LENhave the following sequences:

FR1: DIVMTQS PDSLAVSLGERATINC (SEQ ID NO: 12) FR2: WYQQKPGQPPLLIY(SEQ ID NO: 13) FR3: GVPDRPFGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 14)FR4: FGQGQTKLEIK (SEQ ID NO: 15)

Exemplary heavy chain frameworks of human MAb 21/28′ CL have thefollowing sequences:

FR1: QVQLVQSGAEVKKPQASVKVSCKASQYTFT (SEQ ID NO: 16) FR2: WVRQAPGQRLEWMG(SEQ ID NO: 17) FR3: RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 18)FR4: WGQGTLVTVSS. (SEQ ID NO: 19)These framework sequences are provided for example only; a humanizedantibody can include the human framework region from any humanmonoclonal antibody of interest.

Fragments of the 5 kd106-13 D antibody (or other antibody specific foran epitope of the OPN-5kD fragment), a chimeric form thereof, or ahumanized form thereof are also encompassed by the present disclosure.Antibodies, such as murine monoclonal antibodies, chimeric antibodies,and humanized antibodies, include full length molecules as well asfragments thereof, such as Fab, F(ab′)₂, and Fv which include a heavychain and light chain variable region and are capable of binding theepitope determinant (e.g., amino acids 30-36 of SEQ ID NO:4). In someembodiments, the antibodies fragments have the sequences for V_(L) andV_(H) regions for the 5 kd106-13 D antibody. Fv antibodies are typicallyabout 25 kDa and contain a complete antigen-binding site with three CDRsper each heavy chain and each light chain. To produce these antibodies,the V_(H) and the V_(L) can be expressed from two individual nucleicacid constructs in a host cell. If the V_(H) and the V_(L) are expressednon-contiguously, the chains of the Fv antibody are typically heldtogether by noncovalent interactions. However, these chains tend todissociate upon dilution, so methods have been developed to crosslinkthe chains through glutaraldehyde, intermolecular disulfides, or apeptide linker. Thus, in one example, the Fv can be a disulfidestabilized Fv (dsFv), wherein the heavy chain variable region and thelight chain variable region are chemically linked by disulfide bonds.

In an additional example, the Fv fragments include V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the V_(H) and V_(L) domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which can be subsequently introduced into a host cell such as E.coli to recombinantly express the antibody fragment. The recombinanthost cells synthesize a single polypeptide chain with a linker peptidebridging the two V domains. Methods for producing sFvs are known in theart (see Whitlow et al., Methods: a Companion to Methods in Enzymology,Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Pat. No.4,946,778; and Pack et al., Bio/Technology 11:1271, 1993).

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. Nos. 4,036,945 and 4,331,647,and references contained therein; Nisonhoff et al., Arch. Biochem.Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al.,Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; andColigan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4). Othermethods of cleaving antibodies, such as separation of heavy chains toform monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules. Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Thus, one of skill in the art can readily review the sequences of ananti-OPN-5 kD monoclonal antibody (such as 5 kd106-13 D), locate one ormore of the amino acids listed above, identify a conservativesubstitution, and produce the conservative variant using well-knownmolecular biology techniques. Generally, conservative variants will bindthe target antigen with an equal to or greater efficiency than theparent monoclonal antibody.

Effector molecules, such as therapeutic, diagnostic, or detectionmoieties can be linked to an antibody of interest, such as an antibodythat specifically binds OPN-5kD (e.g., 5 kd106-13 D) using any number ofmeans known to those of skill in the art. In view of the large number ofmethods that have been reported for attaching a variety ofradiodiagnostic compounds, radiotherapeutic compounds, labels (such asenzymes or fluorescent molecules), drugs, toxins, and other agents toantibodies, one skilled in the art will be able to determine a suitablemethod for attaching a given agent to an antibody or fragment thereof.For example, covalent and noncovalent attachment means can be used. Theprocedure for attaching an effector molecule to an antibody variesaccording to the chemical structure of the effector. Polypeptidestypically contain a variety of functional groups; such as carboxylicacid (COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on an antibodyto result in the binding of the effector molecule. Alternatively, theantibody is derivatized to expose or attach additional reactivefunctional groups. The derivatization can involve attachment of any of anumber of linker molecules such as those available from Pierce ChemicalCompany, Rockford, Ill. The linker can be any molecule used to join theantibody to the effector molecule. The linker is capable of formingcovalent bonds to both the antibody and to the effector molecule.Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. Where theantibody and the effector molecule are polypeptides, the linkers may bejoined to the constituent amino acids through their side groups (such asthrough a disulfide linkage to cysteine) or to the alpha carbon aminoand carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will includelinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (forexample when exposed to tumor-associated enzymes or acidic pH) may beused.

An antibody that specifically binds to the OPN-5kD fragment (e.g., 5kd106-13 D or a humanized or chimeric form thereof or a fragmentthereof) can be labeled with one or more detectable agents or labels. Insome embodiments, labels are attached by spacer arms of various lengthsto reduce steric hindrance. Useful detectable agents includeelectron-dense compounds, enzymes, fluorochromes, haptens, andradioisotopes. Anti-OPN-5kD antibodies (e.g., 5 kd106-13 D) can also bedetected using secondary reagents with specificity for mouse IgG. Usefuldetection agents include fluorescent compounds, including fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors, and the cyanine family of dyes (such as Cy-3 or Cy-5) and thelike. Bioluminescent markers are also of use, such as luciferase, Greenfluorescent protein (GFP), Yellow fluorescent protein (YFP). An OPN-5 KDantibody can also be labeled with enzymes that are useful for detection,such as horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, glucose oxidase and the like. When an antibody is labeledwith a detectable enzyme, it can be detected by adding additionalreagents that the enzyme uses to produce a reaction product that can bediscerned. For example, when the agent horseradish peroxidase is presentthe addition of hydrogen peroxide and diaminobenzidine leads to acolored reaction product, which is visually detectable. An antibody mayalso be labeled with biotin, and detected through indirect measurementof avidin or streptavidin binding. The avidin itself can also be labeledwith an enzyme or a fluorescent label. In one example, anOPN-5kD-specific antibody is labeled with electrodense particles, suchas a nanoparticle (for example a gold particle or a semiconductornanocrystal, such as a quantum dot (QDOT®)).

An antibody that specifically binds to the OPN-5kD fragment (e.g., 5kd106-13 D or a humanized or chimeric form thereof or a fragmentthereof) can be labeled with a paramagnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags).

Antibodies can also be labeled with a radiolabeled amino acid, such as³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, or ¹³¹I. The radiolabel maybe used for diagnostic and/or therapeutic purposes. For instance, theradiolabel may be used to detect OPN-5kD expressing cells (such as HCCcells) for example by x-ray or other diagnostic techniques, such aspositron emission tomography (PET) or magnetic resonance imaging (MRI).Further, the radiolabel may be used therapeutically as a toxin fortumors that express OPN (such as HCC).

In some examples a therapeutic agent is linked to an antibody thatspecifically binds to the OPN-5kD fragment (e.g., 5 kd106-13 D or ahumanized or chimeric form thereof or a fragment thereof). Therapeuticagents include various drugs such as vinblastine, daunomycin and thelike, and effector molecules such as cytotoxins for example native ormodified Pseudomonas exotoxin (e.g., see U.S. Pat. Nos. 4,892,827,5,602,095, 5,608,039 and 5,512,658), ricin, abrin (e.g., see Funatsu etal., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol.50:330-335, 1978), botulinum toxins A through F, or Diphtheria toxin(e.g., see U.S. Pat. Nos. 5,792,458 and 5,208,021), ribonucleases (e.g.,see Suzuki et al., Nat. Biotech. 17:265-70, 1999), encapsulating agents,(such as, liposomes) which themselves contain pharmacologicalcompositions, target moieties and ligands. Exemplary toxins are wellknown in the art and many are readily available from commercial sources(for example, Sigma Chemical Company, St. Louis, Mo.). The choice of aparticular therapeutic agent depends on the particular target moleculeor cell and the biological effect desired to be evoked. Thus, forexample, the therapeutic agent may be an effector molecule that iscytotoxic which is used to bring about the death of a particular targetcell. Conversely, where it is merely desired to invoke a non-lethalbiological response, a therapeutic agent can be conjugated to anon-lethal pharmacological agent or a liposome containing a non-lethalpharmacological agent.

The OPN-5kD antibodies or antibody fragments disclosed herein (e.g., 5kd106-13 D) can be derivatized or linked to another molecule (such asanother peptide or protein). In general, the antibodies or portionthereof is derivatized such that the binding to the OPN-5kD fragment isnot affected adversely by the derivatization or labeling. For example,the antibody can be functionally linked (by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody (for example, a bispecificantibody), a detection agent, a pharmaceutical agent, a chemical group(such as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group for example to increase serum half-life or toincrease tissue binding of the antibody) and/or a protein or peptidethat can mediate association of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, such as to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (such as disuccinimidyl suberate). Suchlinkers are available from Pierce Chemical Company, Rockford, Ill.

Nucleic Acid Molecules Encoding Antibodies

Nucleic acid molecules encoding the disclosed OPN-5kD antibodies (e.g.,5 kd106-13 D) or a chimeric or humanized form of any of these antibodiesor a fragment thereof can readily be produced by one of skill in theart. Upon generation of a monoclonal antibody, such as 5 kd106-13 D (seeExample 9), the amino acid sequence can be determined and a nucleic acidsequence encoding the amino acid sequence can be engineered. Inaddition, one of skill can readily construct a variety of clonescontaining functionally equivalent nucleic acid molecules, such asnucleic acid molecules which differ in sequence but which encode thesame effect or molecule (“EM”) or antibody sequence. Thus, nucleic acidsencoding OPN-5kD-specific antibodies (such as those specific for theepitope EELNGAY, amino acids 30 to 36 of SEQ ID NO: 4), conjugates andfusion proteins are provided herein.

Nucleic acid molecules encoding OPN-5kD-specific antibodies can readilybe produced by one of skill in the art, using the amino acid sequencesprovided herein, and the genetic code. In addition, one of skill canreadily construct a variety of clones containing functionally equivalentnucleic acids, such as nucleic acids which differ in sequence but whichencode the same effector molecule or antibody sequence. Thus, nucleicacids encoding OPN-5kD-specific antibodies, conjugates and fusionproteins are provided herein.

Nucleic acid sequences encoding the antibodies that specifically bindOPN-5kD (for example that are specific for the EELNGAY epitope, aminoacids 30 to 36 of SEQ ID NO: 4) can be prepared by any suitable methodincluding, for example, cloning of appropriate sequences or by directchemical synthesis by methods such as the phosphotriester method ofNarang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester methodof Brown et al., Meth. Enzymol. 68:109-151, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,for example, using an automated synthesizer as described in, forexample, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168,1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis produces a single stranded oligonucleotide. This can beconverted into double stranded DNA by hybridization with a complementarysequence, or by polymerization with a DNA polymerase using the singlestrand as a template. One of skill would recognize that while chemicalsynthesis of DNA is generally limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Exemplary nucleic acid molecules encoding sequences encoding an antibodythat specifically binds OPN-5kD (e.g., 5 kd106-13 D) can be prepared bycloning techniques. Examples of appropriate cloning and sequencingtechniques, and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Sambrook et al., supra,Berger and Kimmel (eds.), supra, and Ausubel, supra. Product informationfrom manufacturers of biological reagents and experimental equipmentalso provide useful information. Such manufacturers include the SIGMAChemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.),Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), Invitrogen (San Diego, Calif.), and AppliedBiosystems (Foster City, Calif.), as well as many other commercialsources known to one of skill in the art.

Nucleic acid molecules can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well-known to persons skilled in the art.

In one example, an antibody that specifically binds OPN-5kD (e.g., 5kd106-13 D) is prepared by inserting the cDNA which encodes a variableregion from an antibody into a vector which includes the cDNA encodingan EM, such as an enzyme or label. The insertion is made so that thevariable region and the EM are read in frame so that one continuouspolypeptide is produced. Thus, the encoded polypeptide contains afunctional Fv region and a functional EM region. In one example, cDNAencoding an enzyme is ligated to a scFv so that the enzyme is located atthe carboxyl terminus of the scFv. For example, a cDNA encodinghorseradish peroxidase or alkaline phosphatase, or a polypeptide markerof interest can be ligated to a scFv so that the enzyme (or polypeptidemarker) is located at the amino terminus of the scFv. In anotherexample, the label is located at the amino terminus of the scFv. In afurther example, cDNA encoding the protein or polypeptide marker isligated to a heavy chain variable region of an antibody, so that theenzyme or polypeptide marker is located at the carboxyl terminus of theheavy chain variable region. The heavy chain-variable region cansubsequently be ligated to a light chain variable region of the antibodyusing disulfide bonds. In a yet another example, cDNA encoding an enzymeor a polypeptide marker is ligated to a light chain variable region ofan antibody, so that the enzyme or polypeptide marker is located at thecarboxyl terminus of the light chain variable region. The lightchain-variable region can subsequently be ligated to a heavy chainvariable region of the antibody using disulfide bonds.

Once the nucleic acids encoding the anti-OPN-5kD antibody (e.g., 5kd106-13 D), labeled antibody, or fragment thereof are isolated andcloned, the protein can be expressed in a recombinantly engineered cellsuch as bacteria, plant, yeast, insect and mammalian cells using asuitable expression vector. One or more DNA sequences encoding theantibody or fragment thereof can be expressed in vitro by DNA transferinto a suitable host cell. The cell may be prokaryotic or eukaryotic.The term also includes any progeny of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. Methods ofstable transfer, meaning that the foreign DNA is continuously maintainedin the host, are known in the art. Hybridomas expressing the antibodiesof interest (e.g., a hybridoma that produces 5 kd106-13 D) are alsoencompassed by this disclosure.

Polynucleotide sequences encoding the anti-OPN-5kD antibody (e.g., 5kd106-13 D), labeled antibody, or functional fragment thereof, can beoperatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is ligated suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to appropriate promoters,enhancers, transcription terminators, a start codon (ATG) in front of aprotein-encoding gene, splicing signal for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons.

The polynucleotide sequences encoding the OPN-5kD-specific antibody(e.g., 5 kd106-13 D), labeled antibody, or functional fragment thereofcan be inserted into an expression vector including, but not limited toa plasmid, virus or other vehicle that can be manipulated to allowinsertion or incorporation of sequences and can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide can becarried out by conventional means including preparative chromatographyand immunological separations. Once expressed, the OPN-5kD-specificantibody (e.g., 5 kd106-13 D), labeled antibody or functional fragmentthereof can be purified according to standard procedures of the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y., 1982). Substantially purecompositions of at least about 90 to 95% homogeneity are disclosedherein, and 98 to 99% or more homogeneity can be used for pharmaceuticalpurposes. Once purified, partially or to homogeneity as desired, if tobe used therapeutically, the polypeptides should be substantially freeof endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970, andespecially as described by Buchner et al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5-foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagentN,N′-dicylohexylcarbodimide) are well known in the art.

Use of OPN-5kD Antibodies

The disclosure provides a method for detecting the OPN-5kD fragment in abiological sample, wherein the method includes contacting a biologicalsample with an antibody that binds an OPN-5kD epitope under conditionsconductive to the formation of an immune complex, and detecting theimmune complex, to detect the OPN-5kD in the biological sample. Thedisclosure also provides a method for treating a subject suspected ofhaving an OPN expressing tumor with OPN-5kD specific antibodies, whereinthe method includes contacting a biological sample (in vitro or in vivo)with an antibody that binds an OPN-5kD epitope and inhibits or reducesthe binding of OPN to CD44 receptors (such methods are described hereinand more specifically in the section describing methods of treatment andtherapeutic compositions).

In one example, the detection of the OPN-5kD fragment in the sampleindicates that the subject has a malignancy. In another example, thedetection of OPN-5kD in the sample indicates that a tumor in the subjectis prone to metastasis.

In one example, the antibody that specifically binds OPN-5kD is directlylabeled with a detectable label. In another example, the antibody thatspecifically binds OPN-5 kD (the first antibody) is unlabeled and asecond antibody or other molecule that can bind the antibody thatspecifically binds OPN-5kD is labeled. As is well known to one of skillin the art, a second antibody is chosen that is able to specificallybind the specific species and class of the first antibody. For example,if the first antibody is a human IgG, then the secondary antibody can bean anti-human-IgG. Other molecules that can bind to antibodies include,without limitation, Protein A and Protein G, which are availablecommercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative example, the presence of the OPN-5kD fragment can beassayed in a biological sample by a competition immunoassay utilizingOPN-5kD standards labeled with a detectable substance and an unlabeledantibody that specifically binds an OPN-5kD epitope. In this assay, thebiological sample (such as serum), the labeled OPN-5kD standards and theantibody that specifically binds OPN-5 kD are combined and the amount oflabeled OPN-5kD standard bound to the unlabeled antibody is determined.The amount of OPN-5kD in the biological sample is inversely proportionalto the amount of labeled OPN-5kD standard bound to the antibody thatspecifically binds OPN-5kD.

The immunoassays and methods disclosed herein can be used for a numberof purposes. In one example, the antibodies that specifically bindOPN-5kD are used to detect the OPN-5kD in serum obtained from a subjectknown to have or suspected of having a tumor, such as HCC. Increasedexpression of OPN-5kD is associated with a tumor that expresses OPN,such as HCC, as well as increased likelihood of metastasis of the tumor.Thus, the level of OPN-5kD can be used to diagnose, or determine theprognosis of, HCC in a subject.

EXAMPLE 1 OPN and MMP-9 are Co-Upregulated in HCC

This example describes methods used to demonstrate that OPN and MMP-9are co-upregulated in HCC.

Messenger RNA (mRNA) levels of OPN and MMP-9 were quantified in normaland HCC tissue (obtained from human subjects) by microarray analysis. Asshown in FIG. 1A, expression of both OPN and MMP-9 was significantlyincreased in primary HCC tumors and their corresponding metastaticlesions as compared to non-metastatic tumors (Ye et al., Nat. Med.9:416-23, 2003). As shown in FIG. 1B, up-regulated expression wassignificantly correlated within each patient case (p=0.0013).

EXAMPLE 2 OPN is a Substrate for MMP-9 Activity

This example describes methods used to demonstrate that MMP-9 cleavesOPN into several fragments.

OPN cleavage assays were performed as follows. Human MMP-9 (Calbiochem,CA) was incubated at 37° C. for 3 hours with recombinant human OPN (R&Dsystems, MN) at an optimized ratio (5:1 substrate:enzyme) in a bufferedsolution (200 mM NaCl, 5 mM CaCl, 50 mM Tris-C1, pH 7.5). Digested OPNwas analyzed by 16% tris-glycine SDS-PAGE followed by colloidalcoomassie blue staining (SimplyBlue™, Invitrogen, CA). Separate cleavagereactions (2 μg OPN) were transferred to PDVF membranes for amino-endterminal microsequencing.

For western blot analysis, MMP-9-digested OPN samples and whole celllysates (50 μg) were transferred to 0.22 um PVDF membranes and probedwith a polyclonal antibody generated to a 14-residue peptide justdownstream of the residue 169 thrombin cleavage site (KSKKFRRPDIQYPD;amino acids 169-183 of SEQ ID NO: 2) (Abcam, MA). HEK-293 cell lysateswere probed with an anti-FLAG antibody (Sigma, Mo.). Immuno-reactiveproteins were detected by chemilluminescence (ECL, GE Healthcare LifeSciences, NJ).

Recombinant MMP-9 cleaved OPN at two predominant sites in vitro,residues 166 and 210, resulting in 4 identified fragments: OPN-34 kD(residues 1-166), OPN-32 kD (residues 167-314), OPN-5kD (residues167-210), and OPN-24 kD (residues 211-314). Residue numbering is basedon the sequence shown in SEQ ID NO: 2. Two of the fragments, OPN-32 kDand OPN-5kD, were detected using an antibody that recognizes residues169-183 (FIG. 2A). The OPN-5kD fragment was distinct from the productsof thrombin digestion and could not be detected in cleavage reactionspartially inhibited by the concomitant addition of EDTA, anaturally-occurring inhibitor of MMP-9 (tissue inhibitor of matrixmetalloproteinase-1, or TIMP-1), or the hydroxamate MMP-9 inhibitor I.Cleavage was similarly inhibited by a monoclonal antibody to MMP-9.OPN-5kD formation appeared dependent on further cleavage of OPN-32 kDover time (FIG. 2B).

Expression vectors were generated that included an OPN fragment with itsC-termini fused to a FLAG-tag and its N-termini following the native OPNputative secretory leading 16 amino acid peptide. Complementary DNAsequences encoding the wild-type OPN protein (OPN-a) and threeidentified truncated protein sequences described above (OPN-34 kD,OPN-5kD, and OPN-24 kD) were cloned into a CMV-promoter mammalianexpression vector with a C-terminal 1×FLAG tag (pDest-490) (Gatewaycloning system, Invitrogen, CA). The 16-residue predicted signal peptidesequence (MRIAVICFCLLGITCA, amino acids 1-16 of SEQ ID NO: 2) was fusedto the N-terminus of each construct following a Kozak translationinitiation sequence to ensure similar secretion. Entry clones wereverified by sequencing and gel electrophoresis. As shown in FIG. 3A, thenative 16 amino acid signal sequence (indicated in black) was includedat the N-terminus of each of the 4 sequences. The RGD domain was presentin 2 of the 4 constructs (indicated in white).

Effective expression was demonstrated in HEK 293 cells by immunoblottingfor the C-terminal FLAG tagged sequences as follows. Secreted levelswere also detected in conditioned media samples. HEK 293 cells weretransiently transfected (Lipofectamine 2000, Invitrogen, CA) with 5 μgplasmid DNA and harvested after 24 hours. Total protein cell lysates (50μg) were run on a 4-20% SDS-PAGE gel, transferred to a nitrocellulosemembrane and probed with an anti-FLAG polyclonal antibody. As shown inFIG. 3B, all of the constructs were expressed in HEK 293 cells.

EXAMPLE 3 OPN-5kD and MMP-9 Expression Correlate with MetastaticActivity

This example describes methods used to demonstrate that expression ofthe OPN-5kD fragment (as well as full-length OPN) and expression ofMMP-9 correlated with metastatic potential.

Three human HCC cell lines (Hep3B, SMMC-7721, and MHCC-97) ofestablished inherent differences in invasiveness were comparativelyscreened for OPN protein expression.

Single-cell clones of the low-metastatic HCC cell line SMMC-7721 wereproduced by a limiting dilution cloning technique (Morley et al., Exp.Hematol., 11:418-24, 1983). Briefly, early passage parental cells wereplated in 96-well plates (0.5 cell/well) in supplemented media (20%conditioned media) for two weeks with bi-weekly media changes. Cellswere propagated by sequential splitting to generate large cell culturesfor frozen storage. The subclone most closely resembling the parentalcell line by morphology, growth, and a preliminary evaluation ofadhesion (see assay methods below), SMMC-7721-SC2, was used forsubsequent assays at early passage numbers (<10).

Total protein cell lysates (50 μg) were run by 16% SDS-PAGE, transferredto a nitrocellulose membrane, and probed with a polyclonal OPN antibody(pAb8448) and a monoclonal actin antibody (mAb1501). Recombinant humanOPN and OPN-5kD peptide samples were run as controls (50 ng). As shownin FIG. 4A, endogenous OPN expression corresponds to degree ofmetastatic potential, with the highly-invasive cell line MHCC-97expressing the most prominent levels. Similarly, relative levels of theOPN-5kD cleavage fragment correlated with metastatic potential (FIG. 4A,left panel and FIG. 4C). These results were reproduced using the OPN-5kDspecific polyclonal antibody described in Example 8.

To determine the level of MMP-9 activity in the three human HCC celllines (Hep3B, SMMC-7721, and MHCC-97), gelatinase activities in the celllines were analyzed by zymography of conditioned media samples. Gelatinzymography was performed as follows. Cells were cultured for 48 hours inserum-free media. Cell number-adjusted serum-free conditioned mediaharvested from the HCC cell lines cultured to near-confluence were runon 10% tris-glycine gels containing 0.1% gelatin (NOVEX, Invitrogen,CA). The separated proteins were allowed to renature at 25° C. for 1hour (2.5% Triton X-100) and develop at 37° C. for 16 hours (50 mM Tris,0.2 M NaCl, 5 mM CaCl2, 0.02% Brij 35) followed by staining (0.1% amidoblack). Pro-MMP-9, active MMP-9, and active MMP-2 standards wereincluded as controls (0.5 ug).

As shown in FIG. 4B, the highest levels of gelatinases (pro-MMP-9 andpro-MMP-2) were observed in MHCC-97 cell media. Conversely, the twolow-metastatic cell lines, Hep3B and SMCC-7721, secreted nearlyundetectable MMP-9 levels. These combined findings implicate acorrelated MMP-9/OPN abundance which may confer differential levels ofOPN-5kD.

To measure the invasion of HCC cell lines, the following methods wereused. An equal number of cells (5×10⁴) was seeded into the upperchambers of Matrigel-coated 8-um pore membranes (HTSTM Fluoroblok tumorinvasion system, BD Biosciences). After 36 hours, the invaded cells werelabeled with calcein (37° C., 1 hour) and fluorescence was measured(EX485, EM530). As shown in FIG. 4C, the highest levels of invasion wereobserved in MHCC-97 cells, while the two low-metastatic cell lines,Hep3B and SMCC-7721, had lower levels of invasion.

EXAMPLE 4 OPN Regions Differentially Modulate Cellular Adhesion andMigration

This example describes methods used to demonstrate the effects of OPNfragments on cellular adhesion and migration of HCC cells.

Cellular adhesion was measured using the following methods.SMMC-7721-SC2 cells were seeded into the wells of a 96-wellfluorescent-read plate in serum-free media (6×10⁴ cells/well) with humanOPN (0-250 nM) with or without prior digestion by MMP-9. Media and MMP-9alone treated cells were included as controls. A GRGDS blocking peptide(400 μM) was added to separate media control, intact OPN, and MMP-9cleaved OPN (250 nM) conditions. Non-adherent cells were removed with aPBS wash (100 μl) after one hour and adherent cells were quantified byCyQuant GR dye incorporation (Molecular Probes, OR) and detection atEX485, EM530.

As shown in FIG. 5A, increased SMMC-7721-SC2 adhesion was observed inthe presence of an exogenous MMP-9 cleaved OPN pool versus intact OPNat >50 nM concentrations; addition of an integrin-binding GRGDS peptideblocked both intact and cleaved OPN-mediated adhesion.

To determine the affect of OPN fragments on adhesion, the followingmethods were used. SMMC-7721-SC2 cells (1×10⁶) were trypsinized (E-PET,Biosource, CA) and transfected with the four OPN expression constructsshown in FIG. 3A and empty vector DNA (5 μg) (50-60% efficiency) usingoptimized conditions (Nucleofection, AMAXA, MD) and plated to 6-wellplates. After a 24-hour recovery period, the cells were trypsinized andseeded by equal numbers (6×10⁴) into fluorescent-read 96-well plates inserum-free media. Non-adherent cells were removed with a PBS wash after2-60 minutes and fluorescence was quantified at EX485, EM530.

As shown in FIG. 5B, increased adhesion was observed in response to allfour of the OPN constructs at early time points following transientover-expression (2-10 minutes). However, at later time points (30-60minutes), the OPN fragments containing the RGD integrin binding motif(OPN-full-length and OPN-34 kD) increased adhesion, whereas the non-RGDcontaining regions (OPN-5kD and OPN-24 kD) decreased adhesion relativeto controls (FIG. 5B).

Migration and invasion assays were performed as follows. TransfectedSMMC-7721-SC2 cells (transfected with 5 μg plasmid DNA) were allowed torecover in 5% serum containing media and were re-counted prior todilution in serum-free media. Cells were seeded (5×10⁴) into the upperchambers of Matrigel coated 8 um pore membranes (HTSTM Fluoroblok tumorinvasion system, BD Biosciences, MA) or uncoated 0.8 um pore membranes(HTSTM Fluoroblok insert system, BD Biosciences, MA). Media containingserum (5%) was added to the lower chambers as a chemotactic agent.Blocking antibodies to either the integrin αVβ3 receptor (clone LM609,Chemicon, CA) or a common determinant of the CD44 receptors (clone A020,Calbiochem, CA) were added to the upper chambers (4 μg/ml) at the timeof cell seeding. Migrated cells were labeled with calcein AM (MolecularProbes, OR) at 37° C. for 1 hour, and fluorescence was measured at 24,36, and 48 hours (EX 490 nm, EM 530 nm).

As shown in FIG. 5C, full-length OPN and OPN-34 kD induced SMMC-7721-SC2migration whereas OPN-5kD and OPN-24 kD expression decreased migrationrelative to controls.

EXAMPLE 5 OPN-5kD Induces Cellular Invasion Via CD44, but Not IntegrinαVβ3, Receptors

This example describes methods used to determine the effect of OPNfragments on invasion of HCC cells.

Cell invasion was measured as described in Example 4. Briefly, each ofthe OPN vectors shown in FIG. 3A (5 μg) were separately transfected intoSMMC-7721 cells (1×10⁶) using optimized conditions (Nucleofection,AMAXA). An equal number of cells (5×10⁴) was seeded into the upperchambers of Matrigel-coated 8 um pore membranes (HTSTM Fluoroblok tumorinvasion system, BD Biosciences). After 36 hours, the invaded cells werelabeled with calcein at 37° C. for 1 hour and fluorescence was measured(EX485, EM530).

As shown in FIG. 6A, only the internal OPN-5kD region of OPN inducedreproducible >2-fold increases in cellular invasion in SMMC-7721-SC2cells (p=0.001). Full-length OPN slightly increased invasion, but not tothe extent observed by OPN-5kD. Conversely, over-expression of theRGD-containing OPN-34 kD fragment slightly decreased invasivenesscompared to vector transfected cells at 36 hours which was morenoticeable at a longer 48 hour time point (p=0.005). No significantdifference was observed with OPN-34 kD or OPN-24 kD.

To determine the effect of blocking antibodies to the integrin aV133 andCD44 receptors on the affect of invasion of HCC cells expression OPNfull-length or OPN-5 kD, integrin αVβ3 (Chemicon, CA) or CD44(Calbiochem, CA) blocking antibodies were added (4 μg/ml) to cellstransiently expressing OPN full-length and OPN-5kD. Invading cells weremeasured at 36 hours following calcein incorporation as described above.

To determine the effect of varying concentrations of the OPN-5kD peptideon invasion of HCC cells, SMMC-7721 cells were exposed to increasingconcentrations of the OPN-5kD peptide (0.4 μM, 1 μM and 2 μM) andinvading cells were measured after 36 hours following calceinincorporation as described above. A 44 residue peptide corresponding tothe OPN-5kD fragment:

LRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIPVAQD (SEQ ID NO: 4), wassynthesized and purified >97% by HPLC (EZBiolabs, IN). A GRADSP peptideof no known biological function was tested in parallel as a control.Cells transiently transfected with the OPN-5kD plasmid were included forcomparison.

As shown in FIG. 6B, addition of a blocking antibody that recognizes acommon determinant of CD44 receptors significantly reduced OPN-5kDinduced invasion (p=0.0005), whereas a blocking antibody to the integrinαVβ3 receptor had no significant effect. Similar results were observedwhen a synthetic peptide corresponding to OPN-5kD was used in aconcentration-dependent manner (FIG. 6C).

These results indicate the OPN-5kD peptide induced cellular invasion iscontingent upon CD44 receptor interaction. Similar patterns in adhesive,migratory, and invasive responses were reproduced in the otherlow-metastatic HCC cell line Hep3B.

EXAMPLE 6 Fragments of OPN-5kD reduce OPN-5kD-induced cellular invasion

This example describes methods used to identify a minimum regionresponsible for the observed OPN-5kD-mediated cellular invasion and theidentification of fragments that reduce OPN induced cellular invasion.

A 44 residue peptide corresponding to the OPN-5kD fragment (SEQ ID NO:4), was synthesized and purified >97% by HPLC (EZBiolabs, IN). A seriesof short peptides spanning the region of residues 167-210 of OPN (SEQ IDNO: 2) were generated as follows. Eight small overlapping peptidesspanning the OPN-5kD peptide were similarly synthesized to >75% purity(p1=LRSKSKKFRR, amino acids 167-176 of SEQ ID NO: 2; p2=KKFRRPDIQY,amino acids 172-181 of SEQ ID NO: 2; p3=PDIQYPDATD, amino acids 177-186of SEQ ID NO: 2; p4=PDATDEDITS, amino acids 182-191 of SEQ ID NO: 2;p5=EDITSHMESE, amino acids 187-196 of SEQ ID NO: 2; p6=HMESEELNGA, aminoacids 192-201 of SEQ ID NO: 2; p7=ELNGAYKAIP, amino acids 197-206 of SEQID NO: 2; and p8=YKAIPVAQD amino acids 202-210 of SEQ ID NO: 2)(EZBiolabs, IN). The lyophilized peptides were resuspended in PBS andapplied with cells to the upper wells of Matrigel invasion chambers atequal molar concentrations (2 μM). Invading cells were measured after 36hours following calcein incorporation as described in the examplesabove.

Exogenous treatment of these short peptides to SMMC-7721 SC2 cells atequal molar concentrations did not increase cellular invasion ascompared to OPN-5kD or a control peptide of no known ability to bindcell surface receptors (GRADSP). However, three peptides (p3=PDIQYPDATD,SEQ ID NO: 5; p6=HMESEELNGA, SEQ ID NO: 6; and p7=ELNGAYKAIP, SEQ ID NO:7) had a suppressive effect relative to media alone treated cells(p<0.05) (FIG. 7A).

Upon separate addition of p3, p6, and p7 in conjunction with OPN-5kD(1:1 molar concentrations, 2 μM each), a partial reduction in invasionwas observed (p<0.002) which contrasted the insignificant effect ofanother peptide, p2 (FIG. 7B). The combined addition of p3, p6, and p7completely abolished the OPN-5kD-mediated invasion down to the levels ofmedia controls (p=2×10⁻⁵).

EXAMPLE 7 OPN-c is Highly Expressed in Metastatic HCC

This example describes methods used to identify the OPN splice variantsexpressed in HCC.

Quantitative RT-PCR was performed as follows. RNA was isolated from HCCprimary metastatic tumors (n=17), primary non-metastatic tumors (n=15),and paired non-cancerous tissues collected >2 cm from the lesions, usingTrizol (Invitrogen, CA). Normal liver tissue was used as a referencepool (n=8). Total RNA was quality assessed prior toreverse-transcription (cDNA archive kit, Applied Biosystems, CA).Primers were designed upstream and downstream from unique reportersequences for OPN-a (TCTCCTAGCCCCACAGAATGCTGTG (25-mer, SEQ ID NO: 10),66-bp final amplicon) and OPN-c (AGGAAAAGCAGAATGCTGTGTCCTC (25-mer, SEQID NO: 11), 92-bp final amplicon) (TaqMan gene expression assays,Applied Biosystems, CA). CT threshold values were normalized to 18S rRNA(Applied Biosystems, CA) and data were presented as fold changesrelative to the pooled normal liver samples.

Increased mRNA levels corresponding to both the wild-type OPN-a and thevariant OPN-c were detected in metastatic HCC tumor samples comparedwith non-metastatic HCC (FIG. 8A). This effect was notably greater forOPN-c (p=0.00074 vs. p=0.019 for OPN-a). Further, increased OPN-c levelswere detected in non-cancerous tissue surrounding metastatic lesions butwere not observed for OPN-a, indicating the involvement of thesurrounding stroma in a differential expression of this OPN variant andconditions favoring metastasis (FIG. 8B). A similar difference in OPN-ctranscript levels was reflected in the degree of metastatic potential inthree highly metastatic HCC cell lines evaluated relative to twolow-metastatic cell lines following normalization to primaryhepatocytes.

These results indicate greater mRNA level expression of OPN-c than thewild-type OPN-a in metastatic HCC. The adjacent non-cancerous tissues ofthe metastatic tumor samples are also higher in OPN-c but not OPN-alevels. Histological analysis of this surrounding tissue indicates apredominance of Kupffer-like cells, likely indicating the contributionof macrophages in the tumor cell environment to this observed transcriptlevel response. Thus, these results indicate a macrophage contributionto MMP-9 levels in the tumor cell microenvironment. Such tumor-stromalcell interactions and cross-talk via soluble cytokines and other factorsappear to play a role in malignant cell invasion in a multi-cellulartumor microenvironment.

EXAMPLE 8 Polyclonal Antibody Generation

This example describes methods used to generate polyclonal antibodies tothe OPN-5kD peptide.

Un-conjugated OPN-5kD synthetic peptide (synthesized as described inExample 6) was injected into 2 rabbits (3×500 ug doses), followed by aboost injection (100 μg) at 84 days following initial immunization(Animal Pharm Services, CA). Post-boost bleedings were conducted every 2weeks for 6 months and immuno-specific antibodies were affinity purifiedusing an OPN-5kD peptide column (Affigel 15, Biorad, CA). The resultingIgG antibody was characterized using standard immunoblotting andimmunoprecipitation methods.

EXAMPLE 9 Monoclonal Antibody Generation

This example describes methods used to generate monoclonal antibodies tothe OPN-5kD peptide.

Standard methods were used to produce monoclonal antibodies that werespecific for OPN-5kD (for example see Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). SEQID NO: 4 was injected into mice, followed by boost injections, toproduce monoclonal antibodies. Murine hybridomas were produced.Antibody-producing clones were identified by detection of antibody inthe supernatant fluid by Western blotting. One selected positive clone(5 kd106-13 D) was expanded and the monoclonal antibody productharvested.

Hybridoma 5 kd106-13 D had the highest specificity and sensitivity tobinding the OPN-5kD fragment. As shown in FIG. 9, 5 kd106-13 D antibodydetects the OPN-5 kD fragment with high specificity, and also has somereactivity to full-length OPN. It is thought that full length OPN isbound to the blotting membrane in a manner that partially blocks accessof the 5 kd106-13 D monoclonal antibody to the epitope within theOPN-5kD epitope sequence contained in the full length molecule.

The epitope of the monoclonal antibody produced by hybridoma 5 kd106-13D is EELNGAY (amino acids 30-36 of SEQ ID NO: 4).

EXAMPLE 10 Humanization and Production of an scFv

This example provides methods that can be used to determine the CDRamino acid sequences of the monoclonal antibody 5 kd106-13 D describedin Example 9. CDR amino acid sequences from monoclonal antibody 5kd106-13 D are used for humanization and construction of recombinantscFv and scFv₂ fragments. Protein-based and cell-based assays have beenused extensively for the purpose of evaluating engineered antibodies(reviewed by Qu et al., Methods. 36:84-95, 2005).

Competitive cell-based binding assays are developed to compare theantigen binding capabilities of engineered antibodies with those of theparental mouse monoclonal antibodies. For initial assays, human HCCcells are used as a source of target cells. For these assays, unlabeledengineered antibodies are used as a competitor of antigen binding byphycoerythrin (PE)-labeled parental antibodies. Briefly, human HCC cellsare plated at 1×10⁵ cells/well in a 96-well plate (100 uL/well). Aconstant amount of PE-labeled parental antibody (10 nM) is mixed withvarying concentrations of unlabeled parental or engineered antibodies(0.2-1,000 nM) and added to each well (100 uL/well), with eachexperimental condition set up in triplicate. Plates are preblocked toprevent binding of PE-conjugated antibody to the plate surface(phosphate buffered saline (PBS), 0.05% Tween 20, and 5% fetal calfserum (FCS) for 2 hours at room temperature). After adding cells plusantibodies, the plates are incubated on ice with gentle mixing for 2 hr.Plates are then centrifuged and washed five times to eliminate unboundPE-labeled antibody and evaluated for PE signal using a fluorescenceplate reader. The fluorescence associated with cells is plotted versusthe concentration of unlabeled antibodies, yielding competitiveinhibition curves. Successful engineering results in similar curves forthe engineered and parental antibodies. Competitive radio-immunoassayscan also be used as an alternative for this determination.

EXAMPLE 11 Detection of OPN-5kD Using Antibodies

This example describes methods that can be used to detect the OPN-5kDfragment using OPN-5kD specific antibodies, such as those specific forthe epitope EELNGAY (amino acids 30-36 of SEQ ID NO: 4), for exampleantibodies produced by hybridoma 5 kd106-13 D (see Example 9). Forexample, OPN-5kD can be detected in a biological sample, such as a tumorsample or blood sample (or fraction thereof) to facilitate diseasediagnosis in patients not previously diagnosed with disease,confirmation of diagnosis in patients with tentative diagnosis, ordisease monitoring in patients undergoing treatment for disease. In someexamples, the levels of OPN-5kD are quantified. For example, suchmethods can be used to diagnose the presence of an OPN-expressing tumor,such as HCC or metastatic HCC, provide a prognosis of a subject havingsuch a tumor (for example determine the likelihood that a patient willlikely develop a metastasis), or combinations thereof.

In one example, serum specimens are evaluated from patients with HCC aswell as normal subjects who do not have HCC or other cancer for thepresence of the OPN-5 kD fragment bound by monoclonal antibody 5kd106-13 D (or other monoclonal antibody specific for the epitopeEELNGAY, amino acids 30-36 of SEQ ID NO: 4). Serum specimens areevaluated from 20-50 HCC patients using Western blot analyses, directELISA, or sandwich ELISA. Antibody reactivity with serum specimens fromnormal donors (negative controls) is also assessed. Blood samples (10mL) for preparation of serum is obtained and serum is aliquoted andstored at −80° C. until used. In some examples, serum is also analyzedfor the presence of full-length OPN.

In some examples, full-length OPN (e.g., SEQ ID NO: 2) is removed fromthe serum prior to detection of OPN-5kD, for example using filtration toremove higher molecular weight proteins (e.g., full-length OPN) andallowing lower molecular weight proteins (e.g., OPN-5kD) to passthrough. In some examples there is no fluid dilution factor soconcentration of OPN-5kD in the filtrate is the same as it was in theunfractionated serum. For example, ultrafiltration methods can be usedto physically separate the OPN-5kD fragment from OPN.

In some examples, competition ELISA is used to detect the OPN-5kDfragment in serum using an ultra sensitive electrochemiluminescencedetection system. Competition assays are well-known in the art. In oneexample, the 5 kd106-13 D monoclonal antibody is used as the solid phasecapture reagent and ruthenium-labeled OPN-5kD fragment as theequilibrium detection reagent. Addition of the serum or other testsample competes for the binding to the capture reagent. This results ina reduction of signal as unlabeled OPN-5kD fragment competes with theruthenium-labeled OPN-5kD fragment. Ruthenium is caused tochemiluminesce by electrical current in the microtiter plate (e.g.,provided by Meso Scale Discovery, Gaithersburg, Md.) and the resultantsignal is measured (e.g., by CCD camera).

It is expected that the levels of the OPN-5kD fragment in serum fromnormal volunteers will be significantly decreased relative to patientswith hepatocellular carcinoma (and conversely, the levels of the OPN-5kDfragment in serum from HCC patients will be significantly increased (forexample at least 1.5-fold, at least 2-fold or even at least 5-fold)relative to normal patients), thus demonstrating the utility ofmonoclonal antibodies provided herein to diagnose or prognose anOPN-expressing tumor such as HCC.

EXAMPLE 12 OPN-5kD Specific Antibodies Reduce OPN-5kD-Induced CellularInvasion

This example describes methods that can be used to demonstrate thatantibodies specific for OPN-5kD, such as those described in Examples 8and 9 can inhibit cellular invasion in vitro.

OPN-5kD antibodies (e.g., 5 kd106-13 D or other antibody specific forthe same epitope) are suspended in PBS and applied with cells to theupper wells of Matrigel invasion chambers at equal molar concentrations(2 μM). Invading cells are measured after 36 hours following calceinincorporation as described in the examples above (see Examples 4-6).

Therapeutic antibodies are identified as those that when added at equalmolar amounts with OPN-5kD do not increase cellular invasion as comparedto OPN-5kD (alone) or a control peptide of no known ability to bind cellsurface receptors (GRADSP). Therapeutic antibodies are also identifiedas those that decrease migration as compared to the controls.

EXAMPLE 13 Treatment of HCC in an Animal Model Using OPN5-kD Antibodies

This example describes methods than can be used to demonstrate that atherapeutic composition that includes one or more OPN-5kD specificantibodies can be used to treat HCC (such as metastatic HCC) in ananimal model. One skilled in the art will appreciate that similarmethods can be used to treat other OPN-overexpressing tumors.

Monoclonal OPN-5kD specific antibodies, such as those described inExample 9, for example 5 kd106-13 D or other monoclonal antibodyspecific for the epitope EELNGAY (amino acids 30-36 of SEQ ID NO: 4),will be effective in blocking the growth and invasiveness of HCC cancercells in vivo. The effectiveness of such antibodies in blocking thegrowth of HCC tumors is tested using subcutaneous implantation of HCCtumor cells in mice as a model. The primary advantages of subcutaneousmodels are the ease of implantation and subsequent monitoring of tumorsize. 5×10⁵ HCC cells are inoculated subcutaneously into the rightcraniolateral thorax (axilla) using aseptic technique. Tumors aremeasured every 34 days using vernier calipers until they reach a volumeof 0.2-0.3 cm³. At that time, the mice are divided into four groups(8-10 animals each): Group 1 (vehicle control), Groups 2-4 are treatedwith 0.1, 1.0, or 10 mg/kg a particular test antibody (e.g., 5 kd106-13D), administered intraperitoneally, twice a week. The mice are thenmonitored every 3 days to measure tumor volume (with vernier calipers),body weight, and life span. After no greater than 60 days pastimplantation, the animals are sacrificed and postmortem evaluations oftumorigenesis, including measurement and weight of implanted tumors andproximal lymph nodes, macroscopic evaluation of soft tissues for tumors(lymph nodes and lung), and formalin fixation of the primary tumor andtissues, are performed. The tissues are evaluated byimmunohistochemistry using OPN specific antibodies to determine thelevel of OPN expression in the tumors. In particular, tumor cells thatescape treatment are studied to determine whether they have low levelsof OPN expression.

While subcutaneous implantation is a popular and valuable method formodeling tumor cell growth, differences between the microenvironment ofthe skin and liver can cause rather dramatic differences in cellbehavior. For example, HCC may not metastasize when implantedsubcutaneously whereas intrahepatic implantation (orthotopic) mayfacilitate metastasis. Thus, HCC cells are implanted in the liver byexposing the liver via laparotomy and inoculating tumor cells into liverusing a surgical microscope. After seven days, the mice begin receivingtreatment with antibody, as described above, and the animals aresacrificed no later than 60 days after implantation (or if the animalsbecome moribund). The tumors are palpated at 3-5 day intervals, at whichtime data on tumor size, animal weight, and survival are collected.Post-mortem evaluations are also performed as described above, withemphasis upon the effect of antibody upon metastatic potential (to lungsand regional lymph nodes). The monoclonal antibodies are believed toblock the primary tumor and metastatic potential of HCC cells in adose-dependent manner.

To minimize identification of strain or clonal-specific effects,identical analyses using other model systems can be employed.

One skilled in the art will appreciate other models for liver cancer canbe used, such as s.c. injection of MHCC97 cells with 100% penetrance todevelop lung metastasis in 5 weeks in nude mice (see Ye et al., Nat.Med., 10:416, 2003). OPN-5kD-specific antibodies are then administeredand mice screened as described above. In some examples metastatic tumorcells are administered in the tail-vein injection and lung metastasismonitored.

EXAMPLE 14 Treatment of HCC in an Animal Model Using Fragments ofOPN-5kD

This example describes methods than can be used to demonstrate that atherapeutic composition that includes one or more OPN-5kD peptidefragments, such as a peptide 5 to 60 amino acids in length that has atleast 5 contiguous amino acids of OPN-5kD (e.g., SEQ ID NO: 4), can beused to treat HCC (such as metastatic HCC) in an animal model. Oneskilled in the art will appreciate that similar methods can be used totreat other OPN-expressing tumors.

Mice recognized in the art as a model of HCC can be used. For example,the nude mice model LCI-D20 (see Tang et al., J. Cancer Res. Clin.Oncol. 130:187-96, 2004) or the model described in Ye et al. (Nat. Med.,10:416, 2003) can be used. Mice are administered at least 1 μg of one ormore OPN-5kD peptide fragments 5 to 60 amino acids in length having atleast 5 contiguous amino acids of OPN-5kD (e.g., SEQ ID NO: 4), such asany of SEQ ID NOS: 5-8, intravenously or at least 100 μg (such as 1 mg)intramuscularly. These peptides can be formulated with an inert diluentor with a pharmaceutically acceptable carrier. If desired, othertherapeutic molecules can also be administered, such as one or moreanti-neoplastic agents. The therapeutic compositions can be administeredin a single dose delivery, via continuous delivery over an extended timeperiod, in a repeated administration protocol (for example, by a, daily,weekly, or monthly repeated administration protocol). In one example,mice receive at least weekly doses of the peptide over at least 6months. Control mice can receive no peptide (such as an injection thatonly includes the pharmaceutical carrier).

Following the administration of one or more OPN-5kD peptide fragments,mice are monitored for tumor treatment, such as regression or reductionin metastatic lesions. In particular examples, mice are analyzed one ormore times, starting 7 days following treatment. Mice can be monitoredusing any method known in the art. For example, diagnostic imaging canbe used (such as x-rays, CT scans, MRIs, ultrasound), as well asanalysis of biological samples (for example analysis of blood, tissuebiopsy, or other biological samples), such as analysis of the type ofcells present, or analysis for a particular tumor marker.

A reduction in the number or size of tumors, or a prolonged survivaltime, in the experimental mice, as compared to the control mice,indicates that the one or more OPN-5kD peptide fragments have ananti-neoplastic therapeutic effect.

EXAMPLE 15 Treatment of HCC in Humans with Anti-OPN-5kD or Fragments ofOPN-5kD

This example describes particular methods that can be used to treat aprimary or metastatic HCC tumor in humans by administration of (1) anantibody specific for OPN-5kD (such as 5 kd106-13 D or anothermonoclonal antibody that recognizes the same epitope), (2) one or moreOPN-5kD peptide fragments, such as a peptide 5 to 60 amino acids inlength that has at least 5 contiguous amino acids of OPN-5kD (e.g., SEQID NO: 4), or both (1) and (2). Although particular methods, dosages,and modes of administrations are provided, one skilled in the art willappreciate that variations can be made without substantially affectingthe treatment. One skilled in the art will appreciate that similarmethods can be used to treat other OPN-expressing tumors.

Therefore human patients are treated intravenously with at least 1 μg(such as 1-100 μg) of one or more peptides that include or consist ofthe sequence shown in any of SEQ ID NOS: 5-8, for example for a periodof at least 6 months, at least one year, at least 2 years, or at leastfive years. Administration of the peptides can be used in conjunctionwith normal cancer therapy (for example rather than replacing thetherapy). Thus the therapeutic peptides can be added to the usual andcustomary chemotherapy, surgery and/or radiation treatmentsconventionally used for the particular tumor type, such as HCC.Administration of the therapeutic peptides can be continued afterchemotherapy and radiation therapy was stopped and can be taken longterm (for example over a period of months or years).

Similarly, human patients are treated intravenously with at least 1 μgof 5 kd106-13 D (or a humanized form, chimeric form, or fragmentthereof, or another monoclonal antibody that recognizes the sameepitope), for example for a period of at least 6 months, at least oneyear, at least 2 years, or at least five years. Administration of theantibodies can be used in conjunction with normal cancer therapy (forexample rather than replacing the therapy). Thus the therapeuticantibodies can be added to the usual and customary chemotherapy, surgeryand/or radiation treatments conventionally used for the particular tumortype, such as HCC. Administration of the therapeutic antibodies can becontinued after chemotherapy and radiation therapy was stopped and canbe taken long term (for example over a period of months or years).

Briefly, the method can include screening subjects to determine if theyhave HCC, such as primary or metastatic HCC. Subjects having HCC areselected. In one example, subject having increased levels of the OPN-5kDfragment in their serum are selected. In a clinical trial, half of thesubjects would follow the established protocol for treatment of HCC(such as a normal chemotherapy/radiotherapy/surgery regimen). The otherhalf would follow the established protocol for treatment of the tumor(such as a normal chemotherapy/radiotherapy/surgery regimen) incombination with administration of the therapeutic peptides orantibodies described above. In some examples, the tumor is surgicallyexcised (in whole or part) prior to treatment with the therapeuticpeptides or antibodies.

Screening Subjects

In particular examples, the subject is first screened to determine ifthey have HCC. Examples of methods that can be used to screening for HCCinclude a combination of ultrasound, tissue biopsy, and examination ofalpha-feta protein (AFP) levels, wherein serum AFP levels greater than20 ng/ml or greater than 400 ng/ml and a positive imaging resultindicate that the subject has HCC.

In some examples, the tumor is analyzed to determine if it overexpressesOPN, MMP-9, or both, wherein the presence of such overexpressionindicates that the tumor can be treated with the disclosed therapies.For example serum can be screened for the presence of OPN-5kD, and thetumor can be screened for OPN-c (and in some examples also OPN-a)expression.

However, such pre-screening is not required prior to administration ofthe therapeutic peptides or antibodies described herein.

Pre-Treatment of Subjects

In particular examples, the subject is treated prior to administrationof therapeutic peptides or antibodies described herein. However, suchpre-treatment is not always required, and can be determined by a skilledclinician. For example, the tumor can be surgically excised (in total orin part) prior to administration of one or more therapeutic peptides orantibodies described herein. In addition, the subject can be treatedwith an established protocol for treatment of the particular tumorpresent (such as a normal chemotherapy/radiotherapy regimen).

Administration of Therapeutic Compositions

Administration can be achieved by any method known in the art, such asoral administration, inhalation, or inoculation (such as intramuscular,ip, or subcutaneous). In some examples, the therapeutic compositionincludes one or more of SEQ ID NOS: 5-7 or antibody 5 kd106-13 D (or ahumanized form, chimeric form, or fragment thereof).

The amount of one or more therapeutic peptides or antibodiesadministered is sufficient to treat a subject having HCC. An effectiveamount can being readily determined by one skilled in the art, forexample using routine trials establishing dose response curves. Inaddition, particular exemplary dosages are provided above. Thetherapeutic compositions can be administered in a single dose delivery,via continuous delivery over an extended time period, in a repeatedadministration protocol (for example, by a, daily, weekly, or monthlyrepeated administration protocol).

In one example, therapeutic compositions that include an antibodyspecific for OPN-5kD (such as 5 kd106-13 D or another monoclonalantibody that recognizes the same epitope) or one or more OPN-5kDpeptide fragments, such as a peptide 5 to 60 amino acids in length thathas at least 5 contiguous amino acids of OPN-5kD (e.g., SEQ ID NO: 4)are administered iv to a human. As such, these compositions may beformulated with an inert diluent or with an pharmaceutically acceptablecarrier.

Assessment

Following the administration of one or more therapies, subjects having atumor (for example HCC) can be monitored for tumor treatment, such asregression or reduction in metastatic lesions. In particular examples,subjects are analyzed one or more times, starting 7 days followingtreatment

Subjects can be monitored using any method known in the art. Forexample, diagnostic imaging can be used (such as x-rays, CT scans, MRIs,ultrasound, fiberoptic examination, and laparoscopic examination), aswell as analysis of biological samples from the subject (for exampleanalysis of blood, tissue biopsy, or other biological samples), such asanalysis of the type of cells present, or analysis for a particulartumor marker. In one example, if the subject has a metastatic HCC,assessment can be made using ultrasound, MRI, or CAT scans, analysis ofthe type of cells contained in a tissue biopsy, and AFP levels.

EXAMPLE 16 Evaluation Following Treatment

During or following therapeutic treatment (such as that described inExample 15), subjects can be monitored for the response of theirtumor(s) to the therapeutic peptides or antibodies.

Subjects can receive a complete physical evaluation, CBC, acute care,hepatic and mineral panels and appropriate evaluations of all evaluablelesions (for example by x-ray, MRI, CT scan, ultrasound) are obtainedevery 6-12 weeks during the first six months of therapy and if stable,every 3-6 months thereafter. Other evaluations can be performed asindicated by symptoms or physical findings. For example, surveillance CTof the chest, abdomen and pelvis can be obtained with at least everyother assessment and as indicated by symptoms or physical findings.

EXAMPLE 17 Additional Treatment

In particular examples, if subjects are stable or have a minor, mixed orpartial response to treatment, they can be re-treated afterre-evaluation with the same schedule and preparation of peptide orantibody that they previously received for up to a year of totaltherapy.

A mixed response is the shrinkage of some lesions but an increase inothers. Subjects with mixed responses may only receive treatment for anadditional 2-3 months without showing true disease stability or a bonafide minor or major response (i.e., no further progression). Twore-treatment cycles can be given following a complete response.

EXAMPLE 18 Diagnosis of HCC in Humans

This example describes particular methods that can be used to diagnoseor prognose HCC (such as metastatic HCC) in a human subject. However,one skilled in the art will appreciate that similar methods can be used.In some examples, such diagnosis is performed before treating thesubject (for example as described in Example 15).

Biological samples are obtained from the subject. If blood or a fractionthereof (such as serum) is used 0.1-10 ml of blood is collected. Serumcan either be used directly or fractionated using filter cut-offs toremove high molecular weight proteins (such as full-length OPN). Ifdesired, the serum can be frozen and thawed before use. If a tissuebiopsy sample is used, 1-100 μg of tissue is obtained, for example usinga fine needle aspirate RNA is isolated from the tissue using routinemethods (for example using a commercial kit).

In one example, OPN-5kD protein levels are determined in a serum sampleobtained from the subject. The serum sample described above is incubatedwith the antibody described in Example 9 (5 kd106-13 D) for a timesufficient for the antibody to bind to OPN-5kD in the serum. TheOPN-5kD/antibody complexes are detected, for example using an ELISA.Alternatively, the serum sample is subjected to 16% SDS-PAGE, andtransferred to a membrane (such as nitrocellulose), which is probed withthe 5 kd106-13 D antibody. The antibody/OPN-5kD complexes can bedetected with a secondary labeled antibody, or by observing theappropriated sized protein on the gel. The relative amount ofOPN-5kD/antibody complexes in the serum sample from the subject can becompared to a reference value, such as a relative amount of OPN-5kD/antibody complexes present in a serum sample from a subject nothaving a tumor, wherein the presence of significantly moreOPN-5kD/antibody complexes in the test sample as compared to thereference sample (such as an increase of at least 2-fold, at least3-fold, or at least 5-fold) indicates that the subject has HCC, hasmetastatic HCC, has a poor prognosis, or combinations thereof.

In one example, OPN-c mRNA expression levels are determined in a tumorsample obtained from the subject and in a tissue sample adjacent to (butnot including) the tumor. cDNA is generated from the RNA isolated fromthe tissue samples described above (for example using a commercialreverse transcription kit). OPN-c cDNA is amplified using appropriateprimers (for example using primers having a detectable label), and theresulting OPN-c amplicons detected. The relative amount of OPN-camplicons in the tumor tissue sample can be compared to a referencevalue, such as a relative amount of OPN-c amplicons present in theadjacent non-tumor sample from the subject, wherein the presence ofsignificantly more OPN-c amplicons in the tumor sample as compared tothe non-tumor sample (such as an increase of at least 2-fold, at least3-fold, or at least 5-fold) indicates that the subject has metastaticHCC, has an increased likelihood of a primary HCC metastasizing, has apoor prognosis, or combinations thereof.

In some examples, relative amount of OPN-5kD protein and OPN-c mRNAexpression are determined in the same subject using the methodsdescribed above.

EXAMPLE 19 Screening for Small Molecule Inhibitors

This example provides exemplary methods that can be used to identifysmall molecule inhibitors that mimic inhibition observed with OPN-5kDspecific antibodies.

A binding assay that measures OPN-5kD binding to its antibody (such as 5dk106-13 D), for example an ELISA, Western blot, or other immunoassay.Binding of the antibody to OPN-5kD is detected in the presence andabsence of one or more test compounds. Such test compounds arecommercially available or can be generated using routine methods. Smallmolecules that disrupt the binding between the antibody and the OPN-5kDfragment are candidate inhibitors of OPN-5kD, and thus may be used inthe therapeutic methods provided herein. For example, such molecules maybind to the paratope of the antibody and hence block its ability to bindto the OPN-5 kD fragment. Alternatively, the small molecule inhibitorscan also bind to the OPN-5 kD fragment and block the epitope of theantibody.

Molecules identified using the methods can be further analyzed for theirinhibition of OPN-5kD activity, and the ability to treat anOPN-overexpressing tumor, for example inhibit or reduce metastasis of atumor, for example using the methods described in Examples 12-15.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only examples of the invention and shouldnot be taken as limiting the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

1. A method of diagnosing an osteopontin (OPN)-expressing tumor in asubject, comprising: contacting a tumor sample obtained from the subjectwith an OPN-5kD fragment specific binding agent under conditions whereinan immune complex will form; detecting the OPN-5kD fragment in the tumorsample; and diagnosing the tumor as an OPN-expres sing tumor if theOPN-5kD fragment is detected in the tumor sample.
 2. The method of claim1, wherein the OPN-5kD fragment consists of a peptide sequence of 40 to50 amino acids comprising at least 95% sequence identity to the sequenceshown in SEQ ID NO:
 4. 3. The method of claim 1, further comprisingcomparing the detected OPN-5kD fragment to a control, and wherein an atleast 1.5-fold increase in the detected OPN-5kD fragment in the samplerelative to the control indicates that the subject has an OPN-expressing tumor.
 4. The method of claim 1, wherein the specific binding agentis an antibody, and wherein detecting the OPN-5kD fragment in a samplecomprises: detecting the formation of the immune complex, wherein thepresence of the immune complex detects the presence of the OPN-5kDfragment.
 5. The method of claim 1, wherein the OPN-expressing tumorcomprises hepatocellular carcinoma (HCC).
 6. The method of claim 5,wherein the HCC is a metastasis.
 7. The method of claim 6, wherein themetastasis is an intra-hepatic metastasis.
 8. The method of claim 6,wherein the metastasis is an extra-hepatic metastasis.
 9. A method ofdiagnosing an OPN-expressing tumor in a subject, comprising contacting atumor sample from the subject with an isolated antibody thatspecifically binds to an OPN-5kD fragment epitope sequence EELNGAY(amino acids 30 to 36 of SEQ ID NO: 4); and detecting binding of theantibody to the tumor sample, wherein an increase in the binding of theantibody to the tumor sample as compared to a control indicates that thesubject has an OPN-expressing tumor.
 10. The method of claim 1, whereinthe OPN-5kD fragment consists of the sequence shown in SEQ ID NO:
 4. 11.The method of claim 1, wherein the specific binding agent comprises anantibody.
 12. The method of claim 11, wherein the antibody is apolyclonal antibody.
 13. The method of claim 11, wherein the antibody isa monoclonal antibody.
 14. The method of claim 11, wherein the antibodycomprises a detectable label.
 15. The method of claim 11, wherein theantibody binds to an OPN-5kD fragment epitope sequence EELNGAY (aminoacids 30 to 36 of SEQ ID NO: 4).
 16. The method of claim 1, whereindiagnosing the tumor comprises diagnosing the subject as having anOPN-expressing tumor with metastatic potential, as having anOPN-expressing tumor that has metastasized, as having a poor prognosis,or combinations thereof, if the OPN-5kD fragment is detected in thesample.
 17. The method of claim 1, further comprising comparing thedetected OPN-5kD fragment to a control, and wherein an at least 2-foldincrease in the detected OPN-5kD fragment in the sample relative to thecontrol indicates that the subject has an OPN-expres sing tumor withmetastatic potential, has a tumor that has metastasized, has a poorprognosis, or combinations thereof.